Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.DOI:http://dx.doi.org/10.7554/eLife.08828.001 Mitochondria are structures within cells that provide the energy to power many biological processes that are essential for complex life. These structures are also highly dynamic and go through cycles of fission (in which a single mitochondrion splits in two) and fusion (in which two mitochondria merge into one). These processes both maintain the correct number of mitochondria in a cell and remove damaged ones, and defects in either can result in many diseases.Previous research had shown that mitochondria are in close contact with another cellular structure called the endoplasmic reticulum. The points of contact mark the sites where mitochondria undergo fission, as small tubes of the endoplasmic reticulum wrap around, and then constrict, to split a mitochondrion.Other recent work revealed that a protein called INF2 is anchored on the endoplasmic reticulum where it promotes mitochondrial constriction. This protein builds actin subunits into long filaments that provide the force for constriction. However, it was not clear how INF2 became active, and whether there are proteins on mitochondria that interact with INF2 or actin.Manor, Bartholomew et al. have now used a combination of microscopy-based methods and biochemical analysis to discover that a mitochondrial protein called Spire1C performs all of these roles. Spire1C is found on the outer membrane of mitochondria; it interacts with INF2 to drive the formation of actin filaments that constrict mitochondria. These results suggest that Spire1C bridges the endoplasmic reticulum with the network of actin filaments. Further experiments then showed that increasing Spire1C levels in cells resulted in the mitochondria becoming fragmented due to increased constriction. On the other hand, depleting Spire1C had the opposite effect and caused mitochondria to become unusually elongated. Following on from this work, the next challenge is to see if Spire1C is used differently or similarly in the different processes that involve mitochondrial fission.DOI:http://dx.doi.org/10.7554/eLife.08828.002 Mitochondrial division is a complex process that is essential for survival in mammals  and is fInverted formin 2 (INF2) is a formin family protein that promotes actin filament polymerization in a regulated fashion , 2014. ASpire proteins are membrane-binding actin-nucleators that interact with and regulate formin proteins . SynergiVertebrates have two known Spire genes, Spire1 and Spire2. Each Spire protein contains highly conserved domains with specific capabilities, including: four actin-monomer binding WH2 domains necessary for nucleating actin filaments; an mFYVE domain that binds to intracellular membranes and facilitates oligomerization ; and an We identified and characterized a novel alternate splice-isoform of Spire1 that contains KIND and WH2 domains common to all Spire proteins, as well as a previously uncharacterized unique 58 amino acid alternate exon sequence (ExonC) . After dWhen cells were transfected with a myc-tagged Spire1C construct, the protein showed extensive co-distribution with the mitochondrial marker mitoRFP  also shoTo examine whether Spire1C distributes on the surface or interior of mitochondria we compared the distribution of GFP-ExonC (marking Spire1C) with mitoRFP  using structured illumination microscopy (SIM), which gives a twofold resolution improvement over conventional confocal imaging . GFP-ExoTo confirm Spire1C's mitochondrial outer membrane localization, we employed a fluorescence protease protection (FPP) assay , which cIn support of this notion, transmembrane domain prediction software  indicateWe next performed photobleaching experiments to investigate the dynamics of Spire1C's association with mitochondrial membranes. Photobleaching of a portion of a mitochondrial element that expressed GFP-Spire1C resulted in rapid recovery of fluorescence, with replenishment arising first in regions close to the bleach site and later at regions further away , similarGiven that Spire proteins can nucleate actin via their highly conserved WH2-repeat domain , we nextGiven that actin assembly has been shown to play an important role in regulating mitochondrial fission , the obsSince ER tubules have been implicated in mitochondrial constriction and division , we inveOne way the KIND domain of Spire1C could affect ER-mediated mitochondrial division is by binding to ER-anchored INF2. To test this possibility, we performed in vitro GST pull-down assays. We found that the C-terminal half of INF2 (INF2-CT), but not the N-terminal half (INF2-NT), associated with a GST-tagged Spire1C KIND domain, but not GST alone . AdditioWe next tested whether disrupting Spire1C's interaction with INF2 inhibits mitochondrial fission. To test this hypothesis, we first asked whether removing the KIND domain from Spire1C blocks the increase in mitochondrial division associated with overexpressing a constitutively active INF2 mutant, INF2 A149 , 2014. CMitochondrial fission would be co-dependent on Spire1C and INF2 if Spire1C's interaction with INF2 drives ER-mediated mitochondrial constriction. To test this hypothesis, we employed confocal fluorescence imaging of ER and mitochondria to examine mitochondrial constriction sites in cells co-expressing the ER marker Ii33-mCherry and different variants of Spire1C. Mitochondria constriction was visible at sites of ER-mitochondria crossover slightly more frequently in cells overexpressing Spire1C compared to cells not overexpressing the construct . U2OS cells stably expressing GFP-INF2 was described in Ii33-mCherry was a generous gift from P Satpute . MitoEmerald and mitoRFP were gifts from A Rambold . Spire1C was amplified from mouse brain cDNA using the sequence of NM_194355 as a reference. We expected to obtain a sequence yielding protein corresponding to GI 37595748, however, it contained an additional 58 residues (ExonC). Thorough examination of all available mammalian Spire1 isoforms revealed at least 3 alternatively-spliced exons, which we refer to as exons A (majority of KIND domain), B (protein sequence AVRPLSMSHSFDLS), and C (protein sequence VPRITGVWPRTPFRPLFSTIQTASLLSSHPFEAAMFGVAGAMYYLFERAFTSRWKPSK).To obtain a full-length Spire1C construct, we amplified the mouse Spire1 gene AK129296, which contains the full KIND domain through the first 3 WH2 domains, along with NM_194355, which contains a partial KIND domain and ExonC without Exon B. A series of amplifications of partial gene sequences was then performed to obtain versions of mouse Spire1 that were ± each of exons A, B, and C . Each vaGGCGCGCCATGGAACTGCATACATTTCTGACCAAAATTAAGAGPrimer 1 GCGCTTAATTAATCAGATCTCGTTGATAGTCCGTTCTGAAGPrimer 2 GCGCGGCGCGCCATGGCCAATACCGTGGAGGCTGPrimer 3 GAGCATTAATTAATCTAGTCTGCTCCGTCTAATTTCTTCPrimer 4 GGCGGGCGCGCCATGGCGCAGCCCTCCAGPrimer 5 GACTTAATTAATCTAGTCTGCTCCGTCTAATTTCTTCPrimer 6 GGCGPrimer 7 CCATGTGCTCCAGGAAGAAGCCPrimer 8 CTGCCTTCCAAGCCATACTCTACTCTACPrimers for spire1 gene amplification and plasmid constructionunderlined.All primers are 5′ to 3′. AscI or PacI restriction sites are CGAGGCTGCAGATGAAGGCCCGGAAGATGAAGACGGAGAGAAGAGAAGCATCTCAGCCATCCGGTCCTATCAGGACGTTATGAAGATCTGTGCTGCTCACCTCCCAACTGAGTCGGAGGCACCCAATCATTATCAGGCAGTATGTCGGGCCCTGTTCGCAGAAACCATGGAACTGCATACATTTCTGACCAAAATTAAGAGTGCAAAGGAGAACCTTAAGAAGATTCAAGAAATGGAAAAGGGTGATGAATCTAGCACAGATCTGGAGGACCTGAAAAATGCAGACTGGGCCCGGTTCTGGGTACAAGCGGCGAGGGATTTGCGAAATGGGGTAAAAGCTAAGAAAGTCCAGCAGCGGCAGTACAACCCTCTGCCCATTGAGTACCAACTGACCCCTTATGAGATGGCCGCGGACGACATTCGGTGCAAAAGATACACCGCGAGAAAAGTAATGGTAAATGGTGACGTCCCCCCTCGGTTGAAAAAGAGTGCTCATGAGGTCGCCGCTGACTTTATCAGATCAAGACCCCCTGCAAATCCAGTTTCAGCCAGAAAACTGAAACCAACCCCACCACGGCCACGGAGCCTCCATGAAAGAGCAGCAGAAGAAATTAAAGCAGAAAGAAAGGCTCGGCCTGTGTCACCAGAAGAAATTAGACGGAGCAGACTAGCAGTGCGGCCACTTAGCATGTCTCACAGTTTTGACTTGTCAGATGTCACTACGCCAGAATCTCCAAAGAATGTTGGAGAATCATCTATGGTGAATGGAGGCTTAACATCTCAAACAAAAGAAAATGGGCTGAGCGCTGCCCAGCAGGGGTCMAQPSSPGGEGPQLGAAGGPRDALSLEEILRLYNQPINEEQAWAVCFQCCGSLRAAAARRQPHRRVRSAAQIRVWRDGAVTLAPAAAAAAEGEPPPASGQLGYSHCTETEVIESLGIIIYKALDYGLKENEERELSPPLEQLIDQMANTVEADGSKDEGYEAADEGPEDEDGEKRSISAIRSYQDVMKICAAHLPTESEAPNHYQAVCRALFAETMELHTFLTKIKSAKENLKKIQEMEKGDESSTDLEDLKNADWARFWVQVMRDLRNGVKLKKVQQRQYNPLPIEYQLTPYEMLMDDIRCKRYTLRKVMVNGDVPPRLKKSAHEVILDFIRSRPPLNPVSARKLKPTPPRPRSLHERILEEIKAERKLRPVSPEEIRRSRLAVRPLSMSHSFDLSSPKNVGESSMVNGGLTSQTKENGLSAAQQGSAQRKRLLKAPTLAELDSSDSEEEKSLHKSTSSSSASPSLYEDPVLEAMCSRKKPPKFLPISSTPQPERRQPPQRRHSIEKETPTNVRQFLPPSRQSSRSLDVTTPEVPRITGVWPRTPFRPLFSTIQTASLLSSHPFEAAMFGVAGAMYYLFERAFTSRWKPSKEEFCYPVECLALTVEEVMHIRQVLVKAELEKYQQYKDVYTALKKGKLCFCCRTRRFSFFTWSYTCQFCKRPVCSQCCSDEELQFPKELMEDWSTMEVCVDCKKFISEIISSSRRSLVLANKRARLKRKTQSFYMSSAGPSEYCPSERTINEIKKMRLPSKPYSTLPIFSLGPSALQRGESCSRSEKPSTSHHRPLRSIARFSTKSRSVDKKIND domainWH2 domains Alternate exon BAlternate ExonCSpire boxmFYVE domainSpire1 shRNA constructs were generated by cloning the sequences into Clontech's pSingle-tTS-shRNA vectors using the HindIII/XhoI restriction sites. The sequences cloned into the vector to knockdown Spire1C were 5′-TCGAGGGATTAGACGTAGCAGATTATTCAAGAG ATAATCTGCTACGTCTAATCTTTTTTACGCGTA-3′  and 5′-TCGAGGCGAATAATCTCCTGACTAATTCAAGAGATTAGTCAGGAGATTATTCGTTTTTTACGCGTA-3′ . Oligonucleotides for human INF2 siRNA were previously described in spire1C gene. A mouse tissue cDNA panel  was used as a template for amplification using Primers 7 and 8. Amplified DNA containing ExonC was ∼400 bp, while DNA lacking this exon was ∼200 bp. Multiple oligomer sets were utilized to eliminate non-specific amplification while capturing as many on-target amplifications as possible. PCR reactions were run on a 2% agaorse gel, and bands of the appropriate size were excised from the gel and purified using a QIAquick gel extraction kit . Purified DNA was cloned into the pCR II-TOPO vector using the TOPO-TA cloning kit (Invitrogen). Sequence analysis was used to confirm the sequence of the amplified and inserted DNA.Oligos flanking ExonC were designed to amplify the The Spire1C gene was amplified from mouse cDNA as described above. Vectors used for protein purification include a modified avidin-6x his-MBP-TEV-3x FLAG-Precision construct under the P1 promoter, as well as a modified pGEX vector for N-terminal GST fusion proteins. All vector backbones were gifts of Dr. Aaron Straight and are described in ExonC and the C-terminal 50 residues of mouse Spire1 were cloned into the avi-his-MBP-TEV-3xFLAG-precision vector described above or a modified pGEX vector  using standard techniques . Avi-hisg 4°C. Supernatant was applied to hydrated glutathione resin (2 ml bed volume per liter culture), protein bound for 1 hr at 4°C, and resin was washed extensively with lysis buffer. Protein was eluted with elution buffer  and loaded onto a HiTrapQ anion exchange column. A salt gradient of 150 mM to 1 M was used for protein elution.GST fusion proteins were used for affinity column construction for affinity purification of antibodies. These proteins were purified by using single colonies of transformed Rosetta (DE3) cells to inoculate 400 ml Terrific Broth  cultures containing 100 μg/ml carbenicillin, 34 μg/ml chloramphenicol, which was grown overnight at 37°C. This culture was diluted into 2 l of fresh TB with antibiotics and grown to an O.D. of 0.8–0.9, at which time it was moved to 23°C. After 1 hr at 23°C, cultures were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside for 3–4 hr and harvested as described for purification of avi-his-MBP-TEV-3xFLAG-Precision proteins above. Cells were thawed in lysis buffer , sonicated, and lysates were centrifuged for 30 min at 125,000×Spire1 affinity columns were made using GST-fusion proteins following the method of Two rabbit polyclonal antibodies described above and three commercially available antibodies were used for examining expression patterns of Spire1 protein in various cell types. The antibodies discussed are: (1) Rabbit polyclonal anti-Spire1 C-term (affinity-purified), (2) Rabbit polyclonal anti-Spire1 ExonC (whole antisera), (3) Sigma mouse monoclonal anti-Spire1, (4) Abcam  mouse monoclonal anti Spire1, and (5) Abnova  rabbit antisera to Spire1. Notably, all of the commercially-available antibodies were targeted to residues 482–584 of the Spire1 isoform lacking ExonC (NP_064533), and thus could only detect non-ExonC containing isoforms.Western blots were performed with 5–50 μg cell lysate and antibodies/antisera was tested at various concentrations, temperatures, and lengths of time for best conditions. Optimized conditions for all antibodies used in this work are described below. HRP-conjugated goat anti-rabbit secondary antibody was used at 1:20,000 in all cases.2, 1 mM EGTA, 10 mM Hepes-HCl pH 7.4, 1 mM DTT, 0.02% thesit , 10 μg/ml aprotinin, 2 μg/ml leupeptin, 0.5 mM benzamidine). INF2-CT  and INF2-NT  were purified as described (Spire-KIND (amino acids 1–234) was expressed as a GST fusion protein in bacteria, and purified on glutathione-sepharose  followed by Superdex200 gel filtration (GE Biosciences) of the glutathione-eluted GST-fusion protein. GST-KIND or GST alone was re-bound to glutathione-sepharose in binding buffer  at 23°C for 1 hr before reading fluorescence anisotropy in an M-1000 fluorescence plate reader (Tecan Inc) at 530 nm excitation and 585 nm emission.INF2 C-term  was expressed in bacteria, purified and labeled on its N-terminal amine with tetramethylrhodamine succinimide as described . LabeledCells were washed in phosphate buffered saline  then fixed with 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 for 30 min before being blocked overnight with 4% BSA at 4°C. The next day, cells were incubated with primary antibody for 2 hr, rinsed three times with PBS for 10 min each, then incubated with secondary antibodies (Invitrogen) for 1 hr, rinsed three times with PBS for 10 min each, then counterstained with phalloidin (Invitrogen) for 30 min, then rinsed with PBS three times, then mounted using ProLong Gold antifade reagent.Confocal images were acquired with an Apochromat 63× 1.4 NA objective lens  on a Marinas spinning disk confocal imaging system  using an EM charge-coupled device camera , or a 100× Apo TIRF 1.49 NA objective  on a Yokogawa CSU-X1 spinning disk system using an EM charge-coupled device camera . Cells were imaged in HEPES-buffered growth media. Confocal z-stacks were taken using 200 nm steps. Images were deconvoluted using Slidebook 6. Individual 16-bit tiff image files were exported, then processed using ImageJ.GFP-Spire1C photobleaching experiments presented in SIM imaging of fixed cells was performed using an ELYRA SIM (Carl Zeiss) with an Apochromat 63× 1.4 NA oil objective lens. Five angles of the excitation grid with five phases each were acquired for each channel and each z-plane, which were spaced at 110 nm each. SIM processing was performed using the SIM module of the Zeiss Zen software package. 16-bit grayscale tiffs were subsequently exported to ImageJ for quantification and processing into rendered colored images. Channels in maximum projection images were aligned in the xy-plane using maximum projection images of fluorescent beads.http://rsb.info.nih.gov/ij/plugins/mbf/index.html). When calculating Pearson's values, the mitochondria channel was used as a mask for colocalization. ER-mitochondria intersection sites were visually identified as regions where ER tubules could be clearly visualized crossing mitochondria—these regions were always in the periphery of the cell, significantly restricting the total number of intersections that could be reliably identified. Mitochondria constriction sites were visually identified as regions defined by relative narrowing of mitochondria diameter or reduced fluorescence. Magnifications of boxed regions were generated using ImageJ. Color images of merged 16-bit tiffs exported from the microscope were generated using the ImageJ ‘merge channels’ function. Statistical analysis was performed using Excel . p-values were determined using the unpaired Student's t-test or ANOVA, as appropriate.All image analysis and processing was performed using ImageJ. Mitochondria lengths were measured manually by first setting the scale according to pixel size, drawing a line along the length of the mitochondria, then using ImageJ's ‘measure’ function. Colocalization analysis and rendering was performed using the colocalization plugin included in the MacBiophotonics ImageJ plugin bundle  value r = R, and (ii) the tubule profile remains parallel to the tubule axis. While the tubule length L = 680 nm was required to remain constant during the deformation, the tubule surface area was free to change. This means that the membrane lateral tension was taken to be zero, which guaranteed that the membrane bending energy was the sole contribution to the membrane elastic energy.The boundary conditions for the energy minimization consisted in the requirements that at the tubule left and right edges (i) the tubule cross-sectional radius, The energy minimization and the shape determination for each pressure value were performed using Brakke's ‘Surface Evolver’ program . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom, Pekka Lappalainen, is a member of our Board of Reviewing Editors. One of the three reviewers, Liza Pon, has also agreed to share her identity.Thank you for submitting your work entitled “A mitochondria-anchored isoform of the actin-nucleating Spire protein regulates mitochondrial division” for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Previous studies revealed that constriction of mitochondria, which precedes Drp1-mediated mitochondrial fission, occurs at sites of close contact between ER and mitochondria, and is driven in part by INF2-mediated actin polymerization. Here Manor et al. show that a specific splice-variant of an actin nucleating protein Spire (named Spire1C) localizes to the mitochondria outer membrane through a specific a-helical motif encoded by its alternate exon C. Importantly, they demonstrate that Spire1C promotes actin filament assembly at the mitochondrial surfaces and modulates mitochondrial fission through its actin-binding WH2 domains and its KIND domain, which specifically binds to INF2 formin. Collectively, these studies provide evidence that Spire1C and INF2 cooperate to form mitochondria-ER intersections, and to promote efficient actin filament assembly specifically at these sites.The majority of the data presented in the manuscript are convincing and the study provides fundamentally important new insights into the mechanisms of mitochondrial fission. However, there are few important issues that should be addressed to confirm the conclusions presented and to further strengthen this study.Essentials revisions:1) Most of the studies presented are based on Spire1C over-expression. The authors should thus perform the mitochondrial fission  and ‘mitochondrial constriction’ assays  also on Spire1C knockdown cells. The effects of Spire RNAi should also be verified with an additional shRNAi construct, or the authors could try to rescue the phenotype of Spire RNAi cells. Together, these experiments would perhaps also reconcile some inconsistencies in the data. For example, while cell quantification suggests that Spire1C overexpression induces mitochondrial fragmentation , the images presented in the manuscript  either show normal or even elongated mitochondria.2) The lipid specificity assay  is not particularly convincing and should be omitted from the manuscript. Lipid specificity of a transmembrane protein (or a hydrophobic protein motif that penetrates into the acyl chain region of the bilayer) cannot be studied using lipid strips, where the individual lipid molecules are most likely not organized into a proper bilayer. Thus, if the authors wish to examine lipid specificity of ExonC, they should instead perform proper vesicle co-flotation or co-sedimentation assays.3) The authors should provide better controls for the protease sensitivity assay. This assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM. Furthermore, based on the sequence analysis  ExonC is predicted to consist of two a-helices, which both are long enough (15-20 residues) to span the mitochondrial outer membrane. Would it be possible that these helices could either make a ‘hairpin’, which spans the outer membrane twice, or that this region would instead just ‘horizontally’ penetrate into the acyl-chain region of the mitochondrial outer membrane without spanning the entire bilayer? To provide stronger support for the conclusions presented, the authors should repeat the assay with a C-terminal GFP-fusion of full-length Spire1C (to confirm that the C-terminus of full-length Spire1C indeed does not face the cytosol as proposed in the current version of the manuscript). 1) Most of the studies presented are based on Spire1C over-expression. The authors should thus perform the mitochondrial fission  and ‘mitochondrial constriction’ assays  also on Spire1C knockdown cells. The effects of Spire RNAi should also be verified with an additional shRNAi construct, or the authors could try to rescue the phenotype of Spire RNAi cells. Together, these experiments would perhaps also reconcile some inconsistencies in the data. For example, while cell quantification suggests that Spire1C overexpression induces mitochondrial fragmentation , the images presented in the manuscript  either show normal or even elongated mitochondria.We have performed additional experiments using Spire knockdown cells (using two different shRNA constructs), and added the fission and constriction data to the manuscript , all of which is consistent with our other data.With regards to apparent inconsistencies in mitochondrial phenotype in Spire1C overexpressing cells: We found that in all conditions there was a range of mitochondrial lengths within each cell, as well as between cells. While the average mitochondrial lengths significantly changed depending on Spire or INF2 activities, nearly every cell has some mitochondria that are very short, and at least a couple that are longer. For the sake of clarity in some of our experiments , we used cells with longer mitochondria in order to be able to make measurements as necessary . In order to clarify our data in this regard, we have added as a supplemental figure a histogram showing the distribution of mitochondrial lengths for each condition .2) The lipid specificity assay  is not particularly convincing and should be omitted from the manuscript. Lipid specificity of a transmembrane protein (or a hydrophobic protein motif that penetrates into the acyl chain region of the bilayer) cannot be studied using lipid strips, where the individual lipid molecules are most likely not organized into a proper bilayer. Thus, if the authors wish to examine lipid specificity of ExonC, they should instead perform proper vesicle co-flotation or co-sedimentation assays.We agree that co-flotation and/or co-sedimentation assays would better establish the lipid specificity of ExonC. This will require many more experiments that go beyond the scope of this work. Therefore, we have removed the lipid dot blot and will attempt to establish ExonC’s lipid specificity in a future study.3) The authors should provide better controls for the protease sensitivity assay. This assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM. Furthermore, based on the sequence analysis  ExonC is predicted to consist of two a-helices, which both are long enough (15-20 residues) to span the mitochondrial outer membrane. Would it be possible that these helices could either make a ‘hairpin’, which spans the outer membrane twice, or that this region would instead just ‘horizontally’ penetrate into the acyl-chain region of the mitochondrial outer membrane without spanning the entire bilayer? To provide stronger support for the conclusions presented, the authors should repeat the assay with a C-terminal GFP-fusion of full-length Spire1C (to confirm that the C-terminus of full-length Spire1C indeed does not face the cytosol as proposed in the current version of the manuscript).We think the reviewers may have overlooked our control protein in the protease protection assay in making his/her statement “this assay would benefit from analysis of a protease sensitive IMS protein to confirm that the protease treatment conditions used degrades OM protein without affecting the integrity of the OM.” By definition, if our treatment conditions are not affecting the integrity of the OM, then any IMS protein would not be affected. This is exactly why we used the OMI-mCherry construct as a control in all of our experiments. OMI-mCherry localizes to the IMS – if the OM was being degraded by our digitonin treatment, then OMI-mCherry would escape into the cytoplasm. Similarly, if the OM was degraded enough that trypsin was able to digest proteins in the IMS, then OMI-mCherry would be depleted upon addition of trypsin. In none of our experiments did we observe depletion of OMI-mCherry mitochondrial fluorescence, indicating that the OM was indeed intact in all of our experimental conditions.We agree that it is important to be careful not to make any claims about the C-terminus of Spire1C being in the IMS, since our FPP assay only informs us that the C-terminal region of ExonC (which is in the middle of the full-length Spire protein) is protected from trypsinization. While our FPP assay leaves us confident that the C-terminus of ExonC is protected from the cytoplasm, we were unable to draw any conclusions from these assays as to the localization of the Spire1C C-terminus. While the secondary protein structure prediction software PHYRE identifies two putative alpha-helices within ExonC, only one of those helices was predicted to be a transmembrane domain by predictive software, so we chose not to postulate that there are two transmembrane domains in ExonC. However, we agree that the hairpin model the reviewers have proposed is just as likely to be correct, and is furthermore appealing since this would allow for the C-terminal domain of Spire1C to interact with other cytoplasmic proteins.We agree that an additional FPP assay using a C-terminus GFP-tagged Spire1C construct would ideally clarify the topology of the Spire1C C-terminus. In order to address the reviewers’ comments, we performed an additional FPP assay using a new Spire1C-GFP construct with GFP on the C-terminus. Unfortunately, when we expressed this construct, the protein no longer localized properly to mitochondria, instead displaying a mostly cytoplasmic localization pattern (with barely detectable accumulation on mitochondria). Due to this disrupted localization pattern, we were unable to draw any conclusions from an FPP assay; upon addition of digitonin, all of the GFP fluorescence was almost immediately depleted, even without subsequent addition of trypsin. While this result may give some clue as to how Spire1C localizes to the OM, many more experiments that go beyond the scope of this work would have to be performed in order to determine what this result is telling us. Thus, we have mentioned these latest findings in the text, and modified our interpretation of our FPP data to include the likely possibility that Spire1C’s ExonC localizes in a hairpin or horizontal fashion on the outer leaflet of the outer mitochondrial membrane.


MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5′-fragments were 3′-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5′-3′ degradation or other molecular fates. Some 3′-5′ decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation. HOX B8 mRNA, directed by the perfectly base-matched miR-196, is one of only a few cases of miRNA-directed mRNA decay reported in mammalian cells1011MicroRNAs (miRNAs) are noncoding RNAs that have been shown to posttranscriptionally regulate gene expression and protein synthesis, particularly in mammalian cells, by partially base-pairing to complementary sequences in the 3′-untranslated regions (3′UTRs) of their target mRNAs124et al.It is very important to provide direct evidence for miRNA-mediated target mRNA cleavage and degradation to access the precise mechanism of action in the miRNA-mediated suppression of target gene expression in mammalian cells. The techniques currently used to study the individual or global effects of miRNA action on its target mRNA, such as 5′RACE, ribosome profiling, and RNA-deep sequencing, are powerful and informative2810TUSC2, also known as FUS1), to demonstrate the mammalian miRNA-mediated target mRNA cleavage and regulatory activities in human cells using a novel SLA–RT-PCR assay  could be posttranscriptionally added to the 3′ ends of miRNA-cleaved 5′-mRNA fragments in various organismsragments . These 3ragments  using toragments .TUSC2 target site  that is effective in all let-7 miRNA members. The expression of let-7 miRNAs was depleted in the cells transfected with miR-98 LNAi 48 h after treatment, as shown by qRT-PCR analysis using let-7–specific SL-RT primers  and the 3′ supplementary pairing region, and they share a conserved short stretch of base-paired (7–10 nt) sequences at the seed region  were used as a negative control. The cel-miR-67 was confirmed to have minimal sequence identity with miRNAs in human, mouse, and rat and shown to have no significant biological impact when ectopically expressed in those mammalian cells. Overexpressed miR-622 and let-7d mRNAs were detected by qRT-PCR in H1299 cells transfected with a miR-622 or a let-7 expression vector, respectively  mRNA targeted by miR-142-3p activity in HeLa cells by a U-track–specific SLA–RT-PCR and Sanger sequencing , were detected by agarose gel electrophoresis  promoter, immediately followed by a 3′UTR with two identical copies of let-7 target sequences (T1 and T2) directly derived from TUSC2 mRNA sequences arranged in tandem and a BGH poly(A) signalling sequence  reporter plasmid-based model system with a defined let-7 target and SLA–RT-PCR primer binding sequences and a fully functional mammalian mRNA structure to monitor the precise action of miRNA on its target in a time- and space-dependent manner. The reporter plasmid (pLJ-T214) consisted of an sequence .TUSC2 let-7 target sites were produced: one copy in the 3′UTR of the endogenous TUSC2 mRNA transcript and two copies in the 3′UTR of the exogenously expressed pLJ-T214 EGFP reporter transcript. Endogenous let-7–mediated mRNA cleavage of those target sites would produce 5′-mRNA fragments with identical 3′ termini, which could be detected competitively by the same SL-RT primer in RT. The origins of the cleaved mRNA fragments could be determined by subsequent PCR amplification with either the endogenous or the pLJ-T214 transcript–specific PCR primers, which would generate SLA–RT-PCR amplicons of different sizes. The predicted sizes of the SLA–RT-PCR products derived from target sites T1 and T2 were 240-209 bp and 532-500 bp, respectively, and those derived from the endogenous target site were predicted to be 230-198 bp.In pLJ-T214–transfected H1299 cells, three identical copies of TUSC2 mRNA fragments and internal references for defining the spatial effect of target sites on endogenous let-7 activity by the SLA–RT-PCR assay or SLA-qRT-PCR. Total RNAs from pLJ-T214–transfected H1299 cells at different time points after transfection were examined with the same set of SLA-RT primers used for the endogenous TUSC2 mRNA fragment detection  by SLA-qRT-PCR in the T1 site were consistently stronger than those in its T2 counterpart at any given time; however, intensity patterns at both sites displayed similar trends over the course of transfection. The accumulation patterns of the 3′-uridylated T2 fragments at 16 h after transfection  catalyse the transfer of UMP residues to the 3′ hydroxyl group of RNA and are involved in bulk degradation of histone mRNA in human cells2228A–RT-PCR . Total RA–RT-PCR . The unmA–RT-PCR . The siRe3 genes  showed me3 genes . The 3′-e3 genes . In conte3 genes . No appae3 genes . The oliTUSC2 mRNAs at various sites in partially paired miRNA:mRNA sequences, predominantly within the miRNA seed region or in the 3′ supplementary pairing region. Most of the cleaved mRNA 5′-fragments were oligouridylated at their 3′-termini by TUTase2 in the miRISC, while some 3′-5′ decayed mRNA fragments could also be intercepted by miRISCs stationed on their targets and oligouridylated by other TUTases in the proximity of the miRISC. These 3′-oligouridylated mRNA 5′-fragments accumulated in cytosol and appeared destined for 5′-3′ decay. These findings provide direct evidence for mammalian miRNA-mediated gene silencing by target mRNA recognition, cleavage and 3′-uridylation, leading to degradation of the protein coding sequence–containing 5′-mRNA fragments via a possible bulk 5′-3′ decay pathway.Multiple models have been proposed for the molecular mechanics of miRNA-mediated posttranscriptional repression of target gene expression either by inhibition of mRNA translation, with no apparent impact on mRNA stability, or by initiation of mRNA decay1278307G cap-binding motif has been identified on Argonaute MID domains but was shown to be weak and could not compete with the cap-binding eIF4E to sustain translational suppression33Our findings suggest that the mRNA cleavage and 3′-oligouridylation activities mediated by mammalian miRNAs with partial base-pairing to their target mRNA sequences in Ago2-miRISCs may be a common molecular mechanism for miRNA-targeted gene silencing. We have shown that target gene silencing mediated by let-7 family miRNAs and other human miRNAs could be initiated by Ago2-catalysed endonucleolytic cleavage on base-paired miRNA:mRNA target sites, which is consistent with the fact that the Ago2 endonucleolytic RNase H domain prefers paired bases as substrates15Most of the cleaved 5′ mRNA fragments were oligouridylated at their 3′-termini in the miRISC. Those 5′ mRNA fragments derived from the initial cleavage sites on the same transcript that escaped oligouridine modification in the miRISC could be subjected to the exonucleolytic RNA 3′-5′ decay pathway and would then be intercepted and oligouridylated by other TUTases in proximity to the miRISC. These oligouridylated mRNA fragments could be recognised by the cytoplasmic heptameric Lsm1-7 complex and accumulated in P-bodies2836373839414243Our results provide new insights into the molecular mechanics of mammalian mRNA-mediated gene silencing by showing a cooperative action of miRNA, Ago2/TUTases and other essential molecular components in miRISCs mediating mRNA degradation initiated by mRNA cleavage and concurrent 3′-uridylation. Further studies are needed to understand the precise mechanism of miRNA action in association with specific co-factors in miRISC on posttranscriptional regulation of target gene expression in a complex mammalian cellular system.RNase inhibitor was acquired from New England Bio-Labs (M0314L). DNA primers and RNA oligos were synthesized by Sigma-Aldrich. High-capacity cDNA first-strand synthesis kit, TaqMan Gene Expression Master Mix and TaqMan probes for miRNA quantification were purchased from Applied Biosystems (Life Technologies). The miRCURY LNA inhibitor against hsa-miR-98 was obtained from Exiqon. ExoSAP-IT for PCR Product Cleanup was obtained from Affymetrix. TRIzol LS Reagent, SYBR Green I and 1 Kb-Plus DNA Ladder were obtained from Invitrogen (Life Technologies). Rabbit anti-Ago2 monoclonal antibody was purchased from Cell Signaling Technology. SiGENOME SMARTpool-EIF2C2 anti-Ago2 and ON-TARGET plus non-targeting siRNA as nonspecific controls (siR-NSCs) were purchased from Thermo Scientific.Human H1299 large cell lung cancer and HeLa cervical cancer cells were grown in Roswell Park Both Memorial Institute 1640 medium and Dulbecco modified Eagle medium supplemented with 10% foetal bovine serum (FBS). For transfection, plasmid DNA was adjusted to 0.5 μg/μL and siRNAs (25 μM) were dissolved in sterile water. Plasmid DNA or siRNA was mixed with an equal volume of 8 mM DOTAP:Cholesterol liposome to make DOTAP:Chol:DNA complexes. DOTAP:Cholesterol liposomes were prepared in-house as described previouslySchizosaccharomyces pombe Cid1 enzyme, which showed that TUTase1 and TUTase3 are involved in histone mRNA uridylationmiRNA let-7 knockdown was carried out in H1299 cells transfected with 10 μM of miRCURY LNA anti-miR-98 inhibitor packaged in a DOTAP:Chol:siRNA complex. After 24 h of initial transfection, the medium was refreshed and cultures were returned to incubation for an additional 24 h. Cells were then subjected to direct lysis in TRIzol LS Reagent, and total RNAs were isolated according to the vendor’s recommendation. The let-7 miRNA levels were measured by qRT-PCR assays. Several putative TUTases have been identified in the human genome on the basis of a homology comparison with the U6-terminal transferase and the TUTase mRNA levels were measured by end-point RT-PCR assays as reported by Mullen et al.To determine the effect of TUTase knockdown on miRNA-mediated fragment distribution, we synthesized all TUTase siRNAs and PCR primers according to the ref. RNAs (50 μg) were treated with 10 μL of RNase-free DNase I  and 20 μL of RNase inhibitor in 100 μL of reaction solution for 5 min at 37 °C, 10 min at 80 °C and 15 sec at 95 °C. RT master mixes were prepared using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and aliquots containing 0.25 μg RNA were mixed with 2.5 μL of the appropriate 100 nM SL-RT primers. The reactions were incubated by the following program: 1 min at 20 °C and 10 sec at 37 °C for 60 cycles, 30 min at 37 °C, 20 min at 42 °C, 5 min at 80 °C and hold at 4 °C. The RT products were then subjected to further PCR amplification for end-point PCR evaluation or to real-time PCR for quantitation. Samples not subjected to RT were used as negative controls to avoid any potential DNA contaminants in the RNA preparations.For end-point PCR analyses of cDNA products primed by SLA-RT primers, PCR was performed in TaqMan Gene Expression PCR Master Mix with universal primer and transcript-specific primers. The RT product (1 μL) was mixed in 30 μL of PCR mixtures containing 0.5 μM PCR primers. The PCR program started at 95 °C for 10 min, followed by 30 cycles of 15 sec each at 95 °C, 1 min at 68 °C and 20 sec at 72 °C for an additional 5 cycles, chased with 15 sec at 95 °C, 2.5 min at 68 °C and 2.5 min at 72 °C. PCR products were analysed by electrophoresis on 3% agarose gels in 1× Tris-Boric acid buffer∆Ct method, where normalised ∆Ct = Ct of Sample − Ct of NTC (non-template control). The qRT-PCR conditions for miRNA detection were modified to 10 min at 95 °C, 5 cycles of pre-amplification of 15 sec each at 95 °C, 2.5 min at 61 °C and 2.5 min at 72 °C, then 40 cycles of 15 sec at 95 °C, 1 min at 60 °C and 1 sec at 72 °C. Let-7 family miRNA, hsa-miR-622, hsa-miR-30a and hsa-RNU44 in H1299 cells were determined by SLA–RT-PCR methods. The data were first normalised against hsa-RNU44, and then the changes in miRNA expression level were calculated by the following formula: ∆∆Ct = ∆Ct of miRNA target − ∆Ct of miRNA negative control4546For profiling of relative quantitative mRNA fragment abundance, a real-time SLA-qRT-PCR with ether the TagMan probe-based or SYBR-Green-based method was performed with 1 μL of RT product primed by each SLA-RT primer in triplicate in 40 μL of TaqMan Gene Expression PCR Master Mix with or without containing SYBR Green I, 0.5 μM of universal primer and 0.5 μM of transcript-specific primer. PCR reactions started at 95 °C for 10 min, followed by 10 cycles of pre-amplification of 15 sec at 95 °C, 2.5 min at 68 °C and 2.5 min at 72 °C, then 40 cycles of 15 sec at 95 °C, 1 min at 68 °C and 20 sec at 72 °C on a CFX384 Real-Time PCR Detection System (Bio-Rad). Melting curve analysis was used to confirm a single PCR product in each reaction. RNU44 was measured as a sample control, and RNA fragment relative abundance was calculated by the 2TUSC2 let-7 target site was synthesized by GenScript. A DNA insert was released by BsrGI/BglII and cloned into an EGFP expression cassette as 3′UTR under a CMV promoter. The transcript 3′UTR region on the plasmid was verified by Sanger sequencing. The details of the pLJ-T214 transcript are illustrated in et al.47A DNA fragment with two copies of let-7 targets identical to the EGFP open reading frame        stop codonTGTACAAGTACGGCATGGACGAGCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAATTAGCATTTCAGCCGTTTGCTACCTCGATTCCTCCCTAGTCAGGCTAGCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCCTGATTCTTCCGACCATTTGTTCCCGCCTTCAGATGCTCGAGAGCATTTCAGCCGTTTGCTACCTCGATTCCTCCAGATCTCTAGTCAGGATAAACCCGCTGATCAGCCTCGA-BGH poly(A) Signal.TUSC2 let-7 target site was synthesized by GenScript. A DNA insert was released by BsrGI/BglII and cloned into an eGFP expression cassette as 3′UTR under a CMV promoter. The transcript 3′UTR region on the plasmid was verified by Sanger sequencing. The details of the pLJ-T722 transcript are illustrated in A DNA fragment with two copies of let-7 targets identical to the EGFP open reading frame        stop codonTGTACAAGTACGGCATGGACGAGCAAGCGGCCGCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCATTTCAGCCGTTTGCTACCTCGGGACGAGGTGCCTAAAGGACTGACCGGCAAGTTGGACGCCCGCAAGATCCCCATAACCTTCAGCTGTGAGCGAAGAAACTCCCAGGCTCAATCAAGGTGTGGCTTCCATTGAGGAGCCCAGGCTGCCACAACCCTGAATAAACTCTGTTGGCGGTCGGTCCACAGTATTGGTTGGTGTTGGTTTGTGTGTGGACAAGAGGTAGTCAGAGCCTGATTCTTCCGACCTACCTGCCATTTGTTCCCGCCTTCAGATGCTCGAGAGCATTTCAGCCGTTTGCTACCTCGTAGATCTGATAAACCCGCTGATCAGCCTCGA-BGH poly(A) Signal.PCR was performed on genomic DNA extracted from normal human bronchial epithelial cells with the following primers flanking the miR-622 precursor:GGACTAGTGAATTCTTTAGAGAAGCTGGACAAGTACTACTTGTAACTCGAGAAAAGTGTCATCTCAGCAGCTCSpeI/EcoRI and cloned into a CMV promoter–driven expression plasmid with BGH poly(A) signal. The plasmid expression cassette was verified by Sanger sequencing.PCR fragments were digested with CMV promoter–driven expression plasmid with BGH poly(A) signal was digested by EcoRI/XhoI and purified as a cloning vector. The correct clones were screened and verified by Sanger sequencing as a let-7d expression vector.The following oligonucleotides were synthesized by Sigma-Aldrich and annealed as inserts. A AATTCCCTAGGAAGAGGTAGTAGGTTGCATAGTTTTAGGGCAGGGATTTTGCCCACAAGGAGGTAACTATACGACCTGCTGCCTTTCTTAGGCTCGAGCCTAAGAAAGGCAGCAGGTCGTATAGTTACCTCCTTGTGGGCAAAATCCCTGCCCTAAAACTATGCAACCTACTACCTCTTCCTAGGGet al.TUSC2 5′-mRNA fragment detection and quantification and nU-track detection, and the TUTase-siRNAs for each experiment, are summarised in SLA-RT primer was composed of stem loop and probe as reported by Chen TUSC2 mRNA fragments with various uridine tails were detected by qRT-PCR assay. The A4 RNA fragments of pLJ-T214 transcripts were represented by 528-bp and 234-bp amplicons and detectable in both unmodified and oligouridylated forms in total RNA samples from H1299 cells transfected with plasmid pLJ-T214. The A4 RNA fragments were further tested with SL-RT primers with different probes to confirm the oligouridine detection methods. Total RNAs from pLJ-T214-transfected H1299 cells were reverse-transcribed with 5 SL-RT primers with different probe compositions to compare their priming capabilities, and the results are shown in 18RNA fragment abundance varies with oligouridine lengths see . Let-7–mHow to cite this article: Xu, K. et al. MicroRNA-mediated target mRNA cleavage and 3′-uridylation in human cells. Sci. Rep.6, 30242; doi: 10.1038/srep30242 (2016).


Understanding the quantitative functional consequences of human disease mutations requires silencing of endogenous genes and expression of mutants at close to physiological levels. Changing protein levels above or below these levels is also important for system perturbation and modelling. Fast design optimization demands flexible interchangeable cassettes for endogenous gene silencing and tuneable expression. Here, we introduce ‘TEMTAC’, a multigene recombineering and delivery system for simultaneous siRNA-based knockdown and regulated mutant (or other variant) expression with different dynamic ranges. We show its applicability by confirming known phenotypic effects for selected mutations for BRAF, HRAS, and SHP2. Recent work provided evidence that quantitative rather than qualitative differences in the proteomic inventory between different cell types and tissues are the cause of functional differences123456Gene silencing based on short interfering RNAs (siRNA) has been demonstrated in several organisms, including in cultured mammalian cells11in vitro, optionally in high-throughput by robotics15Plasmid-based gene delivery systems have become essential for molecular and cell biology. However, the number of available selection markers and the physical space that needs to be available to allow entry of multiple plasmids into cells are technically limiting. These problems are exacerbated by imbalanced delivery if multiple plasmids are used, resulting in heterogeneous cell populations which interferes with read-out. These impediments can be circumvented by combining independent modules containing genes of interest, regulatory elements and other desired functionalities into a single multifunctional multigene delivery plasmid. This is a viable option if the combination of the modules is sufficiently straight-forward. Addressing this challenge, we previously developed the ‘ACEMBL’ system to enable fast and flexible generation of multigene delivery constructs by an automatable technique called tandem recombineering (TR) to combine ‘Donor’ and ‘Acceptor’ plasmid modules via reversible Cre-loxP fusion Tunable Endogenous Mammalian TArget Complementation by multiplexed plasmid-based recombineering. Retaining the original ACEMBL concept, we developed this novel system which affords the means to simultaneously knockdown endogenous targets via RNA interference89in vitro exploiting the Cre-Lox recombination reaction, which can be carried out optionally in high-throughput by robotics applying automated routines we have implementedHere, we introduce TEMTAC, a novel system for http://www.invivogen.com/sirna-wizard). Donor D1 containing the shRNAs was transiently transfected in HEK293 or HeLa cells. After 24 and 48 hours, cells were lysed and endogenous protein levels detected by Western blotting, real time PCR, and deep sequencing. We observed the best silencing effect 48 h post transfection and no  down-regulation using random (‘scrambled’ scrb) shRNAs as a control  contains the silencing cassette for knocking down the endogenous gene by RNA interference . In one 192021 control . IncomplE. coli) mutant and three VP16 (transcription activation domain of herpes simplex virus). In the presence of the inducer doxycycline (dox), the TETon3G-dox complex binds to the TRE minimal promoter (TRE-pMCVmin) and induces the expression of the gene of interest (GOI).Donor D2.1 (pMDS-GOI-TETon3G), constitutively expresses the TETon3G transactivator , a fusion protein of a Tet repressor (Donor D2.2 (pMDS-GOI-TEToff) is based on the TEToff advanced inducible expression system (Clontech). This vector expresses the TEToff advanced transactivator (TetR) and also contains a TRE-based module that expresses high levels of the GOI in the absence of dox.Donor D2.3 (pMDS-GOI-TETon-PGK), is a modification of Donor D2.1, where the expression of the rtTA is downstream of a minimal phosphoglycerate kinase (PGK) promoter .Donor D2.4 (pMDS-GOI-TETon-tTS) is a modification of TETon to reduce basal expression levels. This is achieved by adding an internal ribosome entry site (IRES) and a tTS , which is a fusion of the TetR and the KRAB-AB silencing domain of the Kid-1 protein, after the transactivator (rtTA). As tTS competes with rtTA by binding specifically to the TRE, it suppresses the transcription in the absence of dox.Achieving expression levels that are close to endogenous levels is a crucial prerequisite for analysing the physiological effect of mutations. This is particularly important when studying signalling proteins because of their frequent involvement in downstream activation and feedback loops, which often function at very precise protein levels that need to be maintained meticulously. A major problem in many TET regulated systems is basal expression (without the inducer being present), especially for proteins that are present at markedly low abundance in cells. Furthermore, depending on the protein of interest, different dynamic expression ranges of mutants would be highly desirable to properly recapitulate physiological events. To address this, we created as Donor D2 variants four different TET-related expression system versions with defined properties  in HEK293 cells grown in serum-containing medium. Exogenous expression of this oncogenic mutation tends to be higher as compared to wildtype (WT) . To rectify this, we took advantage of our TET-regulated system to tune the expression levels of WT and V600E to be close to identical  and Donor D2 (pMDS-GOI) were created based on the original plasmids of the ACEMBL system for protein complex expression in prokaryotic hostsde novo from scratch. All DNA elements . Sequences are provided in the shRNAs and genes of interest (GOI) were inserted into the TEMTAC system using standard restriction-ligation cloning methods or, alternatively, by Gibson cloningHEK293, HeLa and GH-HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with L-glutamine and 10% (v/v) heat-inactivated TET-free fetal calf serum (Clontech ref. 631106) .HEK293 or HeLa cells were seeded in 96-well-plates (Thermo Scientific 165306) and grown to 60% confluency in normal growth medium. Cells were transfected with 40 ng of the four different Donor D2 plasmids containing luciferase as the GOI (Donor D2.1-D2.4) or with the fully Cre-assembled (A-D1-D2) pTEMTAC composite plasmid (containing siRNA scramble) using lipofectamine 2000 . 24 hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich). 24 hour later the luciferase assay was performed according to the manufactures instructions .HEK293 cells were seeded on 6 well plates and grown to 60% confluency in normal growth medium. Cells were transfected with 2.5 μg of the fully Cre-assembled A-D1-D2 composite plasmid containing mCherry as the GOI (containing siRNA scramble) using lipofectamine 2000 (Invitrogen). 24 hours after transfection, cells were supplemented with indicated amounts of doxycycline . 24 h later YFP and mCherry fluorescence was imaged on an inverted DMI-6000 Leica wide-field fluorescent microscope equipped with a Leica DFC 350FX camera with a 20× objective.GH-HEK293 cells  were seeded on 6-cm dishes in normal growth medium, grown to 60% confluency, and transfected with 2.5 μg of pTEMTAC (A-D1-D2) plasmid  or, alternatively, pMDC-RNAiDual (shRNA against SHP2 or scramble shRNA) plasmid, using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 24 hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich) and incubated in minimal medium (DMEM plus 2 mM glutamine and 1% serum). 24 hours after transfection, cells were stimulated with 125 ng/ml growth hormone for 10 minutes, washed with PBS and resuspended in 200 μl of lysis buffer . Lysed cells were analysed by western blot.HEK293 were seeded on 6-cm dishes in normal growth medium, grown to 60% confluency, and transfected with 2.5 μg of pTEMTAC (containing BRAF/HRAS WT or V600E/G12V as GOI) or pMDC-RNAiDual (shRNA against BRAF/HRAS or scramble shRNA) plasmid, using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 48 hours after transfection, cells were supplemented with indicated amounts of doxycycline (Sigma-Aldrich) and incubated in normal growth medium. 24 hours after transfection, cells were washed with PBS and resuspended in 200 μl of lysis buffer . Lysed cells were analysed by western blot.Cell lysates were loaded on SDS gels and separated by electrophoresis; gels were then transferred onto nitrocellulose membrane using the iBlot system (Invitrogen), and the blots were incubated 1 h at room temperature in TBS, Tween 0.1%+5% milk. The primary antibody was incubated at 4 °C overnight (1:1000 dilution), and the HRP-coupled secondary antibody (1:10 000 dilution) was incubated 1 h at room temperature, both in TBS Tween 0.1% + 0.5% milk. Blots were developed using high sensitivity ECL reagent (Thermo) and visualized using the Fujifilm LAS-3000 developer. Bands were analyzed using ImageJ. The following antibodies were used for Western blotting: SHP2 , BRAF , total RAS , growth hormone receptor , phospho-ERK Thr202/Tyr204 , phospho-MEK Ser217 and Ser221  and β-actin .® Green RNA-to-CTTM 1-Step Kit, Applied Biosystems), 0.15 μM of forward and reverse primers and 60 ng of RNA following the manufactures instructions. For PCR the cycling parameters were (i) reverse transcription , (ii) activation of polymerase and initial denaturation , (iii) 40 cycles of denaturation , annealing, extension and read fluorescence , (iv) final hold (4 °C). The following primers were used. For endogenous BRAF 5′ATCCCAGAGTGCTGTGCTGT3′ (forward) and 5′TCTCCAACACTTCCACATGC3′ (reverse), for exogenous BRAF 5′AATGTTGCGCCGTTTACAG3′ (forward) and 5′TCTCCAACACTTCCACATGC3′ (reverse), for endogenous HRAS 5′TGCCATCAACAACACCAAGT3′(forward) and 5′ACGTCATCCGAGTCCTTCAC3′ (reverse), for exogenous HRAS 5′GAGGGCTTCCTGTGTGTCTT3′ (forward) and 5′TCTTTGACGCGCTTAATTTG3′ (reverse), for endogenous SHP2 5′CTTGTACTCCAACGCCACCC3′ (forward) and 5′CTGTGCTGAAGTTTTGGCAGG3′ (reverse) and for total SHP2 5′GGTGTGGAGGCAGAAAACCT3′ (forward) and 5′TTGATGTGGGTGACAGCTCC3′(reverse).Cells were grown and transfected as before. RNA was isolated using miRNeasy Mini Kit (Qiagen) following the manufactures protocol including the optional DNase digestion step. PCR reactions were prepared using qPCR mix (Power SYBRTM II RT (Invitrogen) with random oligonucleotides. In the first PCR all target genes  were amplified for 5 cycles using Phusion DNA polymerase (New England Biolabs) with the following degenerated adaptor primers: for BRAF (region of V600E)RNA was extracted as described for real time PCR. cDNA synthesis was done using SuperScript5′CCCTACACGACGCTCTTCCGATCTCTTCATGAAGACCTCACAG3′5′CCCTACACGACGCTCTTCCGATCTaCTTCATGAAGACCTCACAG3′5′CCCTACACGACGCTCTTCCGATCTgaCTTCATGAAGACCTCACAG3′5′CCCTACACGACGCTCTTCCGATCTcgtCTTCATGAAGACCTCACAG3′5′CCCTACACGACGCTCTTCCGATCTagatCTTCATGAAGACCTCACAG3′(forward) and5′TTCAGACGTGTGCTCTTCCGATCTCTGTTCAAACTGATGGGACC3′5′TTCAGACGTGTGCTCTTCCGATCTaCTGTTCAAACTGATGGGACC3′5′TTCAGACGTGTGCTCTTCCGATCTgaCTGTTCAAACTGATGGGACC3′5′TTCAGACGTGTGCTCTTCCGATCTcgtCTGTTCAAACTGATGGGACC3′5′TTCAGACGTGTGCTCTTCCGATCTagatCTGTTCAAACTGATGGGACC3′(reverse), for HRAS (region around G12V)5′CCCTACACGACGCTCTTCCGATCTGACGGAATATAAGCTGGTGG3′5′CCCTACACGACGCTCTTCCGATCTaGACGGAATATAAGCTGGTGG3′5′CCCTACACGACGCTCTTCCGATCTcaGACGGAATATAAGCTGGTGG3′5′CCCTACACGACGCTCTTCCGATCTgctGACGGAATATAAGCTGGTGG3′5′CCCTACACGACGCTCTTCCGATCTatcaGACGGAATATAAGCTGGTGG3′(forward) and5′TTCAGACGTGTGCTCTTCCGATCTGTCGTATTCGTCCACAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTaGTCGTATTCGTCCACAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTcaGTCGTATTCGTCCACAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTatcGTCGTATTCGTCCACAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTatcaGTCGTATTCGTCCACAAAGTG3′(reverse), for SHP2 (region around D61G)5′CCCTACACGACGCTCTTCCGATCTAGTAAAAGTAACCCTGGAGAC3′5′CCCTACACGACGCTCTTCCGATCTaAGTAAAAGTAACCCTGGAGAC3′5′CCCTACACGACGCTCTTCCGATCTgaAGTAAAAGTAACCCTGGAGAC3′5′CCCTACACGACGCTCTTCCGATCTtgaAGTAAAAGTAACCCTGGAGAC3′5′CCCTACACGACGCTCTTCCGATCTctgaAGTAAAAGTAACCCTGGAGAC3′(forward) and5′TTCAGACGTGTGCTCTTCCGATCTGACCAACTCAGCCAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTaGACCAACTCAGCCAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTgaGACCAACTCAGCCAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTtgaGACCAACTCAGCCAAAGTG3′5′TTCAGACGTGTGCTCTTCCGATCTctgaGACCAACTCAGCCAAAGTG3′(reverse), and for SHP2 (region around C459G)5′CCCTACACGACGCTCTTCCGATCTGAGGTGCACCATAAGCAGGAG3′5′CCCTACACGACGCTCTTCCGATCTaGAGGTGCACCATAAGCAGGAG3′5′CCCTACACGACGCTCTTCCGATCTgaGAGGTGCACCATAAGCAGGAG3′5′CCCTACACGACGCTCTTCCGATCTtgaGAGGTGCACCATAAGCAGGAG3′5’CCCTACACGACGCTCTTCCGATCTctgaGAGGTGCACCATAAGCAGGAG3′(forward) and5′TTCAGACGTGTGCTCTTCCGATCTTCACAATGAACGTCCCTGTC3′5′TTCAGACGTGTGCTCTTCCGATCTaTCACAATGAACGTCCCTGTC3′5′TTCAGACGTGTGCTCTTCCGATCTgaTCACAATGAACGTCCCTGTC3′5′TTCAGACGTGTGCTCTTCCGATCTtgaTCACAATGAACGTCCCTGTC3′5′TTCAGACGTGTGCTCTTCCGATCTctgaTCACAATGAACGTCCCTGTC3′(reverse).The resulting amplicons were used for determination of cycle numbers for the second PCR with the TruSeq dual barcoding primers. The primary PCR product was amplified using NEBNext 2x High Fidelity PCR Master Mix (New England Biolabs), with 200nM primer concentration, and 0.1x SYBR Green I  monitoring the amplification in a Roche LightCycler 480. The cycle number for the final library PCR was chosen to get a sufficient amount of product without reaching the plateau phase of the PCR. Then 2.5 μl of the primary PCR product was used for amplification with the NEBNext Master Mix and barcode primers  but without SYBR Green, for the number of cycles determined from the qPCR. PCR products were purified using AMPure XP beads and concentration was determined on a Bioanalyzer DNA 1000 chip. Samples were sequenced using MiSeq v3 chemistry with 130 cycle single reads in a MiSeq sequencer (Illumina). Flanking reads were subtracted by mutant reads to calculate the reads corresponding to the endogenous expression level.How to cite this article: Beltran-Sastre, V. et al. Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering. Sci. Rep.5, 17432; doi: 10.1038/srep17432 (2015).


MAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1° sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.HIV-2 and SIV MAC/SIVSM lineage, with restriction being greatest for SIVSM and the least for epidemic HIV-2. Here we show that this dominant-acting, antiviral activity is specific for the capsid and blocks the virus after it enters the nucleus. The evidence suggests that, in order to jump from sooty mangabey monkeys to humans, the capsid of these viruses changed in order to adapt to this antiviral activity. In keeping with the practice concerning anti-lentiviral activities we propose to call this new antiviral activity Lv4.HIV-1 and HIV-2, the two lentiviruses that cause AIDS in humans, are members of a family of such viruses that infect African primates. HIV-1 is a zoonosis that was transmitted to humans from chimpanzees. HIV-2 was transmitted to humans from sooty mangabey monkeys. In several documented cases of cross-species transmission of lentiviruses it has been shown that replication of the virus in the new host species necessitated that the virus adapt to species-specific antiviral factors in the host. Here we report that human blood cells possess an antiviral activity that exhibits specificity for viruses of the HIV-2/SIV CPZ), and perhaps from gorillas (SIVGOR) [CPZ itself is probably a recombinant virus that resulted from co-infection of a chimp with viruses transmitted from a red-capped mangabey (SIVRCM) and a greater spot-nosed monkey (SIVGSN) [CPZ did not cause disease in chimpanzees but extensive observation of feral animals has demonstrated that this is not the case [Human immunodeficiency virus type 1 (HIV-1) is the major cause of the acquired immune deficiency syndrome (AIDS) pandemic. Among the immunodeficiency viruses that infect at least 40 of the primate species in sub-Saharan Africa, the simian immunodeficiency viruses (SIVs) found in central African chimpanzees and gorillas are monophyletic with HIV-1 . Each of(SIVGOR) –6. SIVCP(SIVGSN) . Until rthe case .Cercocebus atys) on multiple occasions [SM, but cross-species transmission to another non-native host, rhesus macaques (SIVMAC), resulted in AIDS [HIV-2, a second AIDS-causing virus that has highest prevalence in West Africa, was transmitted to people from sooty mangabey monkeys  family can disrupt retroviral replication in a species-dependent manner [Though transmission of primate lentiviruses to humans has occurred on multiple occasions and may still be occurring , these et manner –18. TRIMt manner . Moreovet manner ,21 and it manner . TRIM5α t manner  and has t manner . Inhibitt manner –27. Addit manner –30. Finat manner ,30–32.MAC and SIV from African green monkeys (SIVAGM) [HIV-1 is inhibited by TRIM5α from a number of African and Asian monkey species, such as rhesus macaques, African green monkeys, and sooty mangabeys ,33. The (SIVAGM) ,36–39.MAC replication are not inhibited by TRIM5α in human cells. These experiments, however, all used immortalized adherent cell lines such as TE671 (rhabdomyosarcoma) [MAC or HIV-1 in a range of mammalian cell lines [MAC vectors in several human cell lines, e.g. Raji (B lymphocyte) and in the T lymphocyte cell lines Jurkat, HuT78 and CEM. This raised the possibility that lentiviruses could be inhibited in a cell-type specific fashion in human cells. In the work presented here, we investigated restriction to SIVMAC replication in peripheral blood lymphocytes (PBLs) as well as in various cell lines. Our data reveal a TRIM5α-independent restriction activity targeting SIVMAC, and the related SIVSM and HIV-2, in human blood cells.Thus, available data suggest that the early post-entry stages of SIVsarcoma) ,40,41, Hsarcoma)  or HeLa sarcoma) ,31,43. Hll lines . They foNL4-3 and SIVMAC239, as previously described [nef was replaced with GFP coding sequence, such that the fluorescent reporter was expressed from the respective LTR. The two vectors were produced in parallel by collecting supernatant from transfected 293T cells. The vector-containing supernatants were checked for reverse transcriptase activity [Human cell lines were challenged with VSV G-pseudotyped, single-cycle vectors derived from HIV-1escribed . In eachactivity , normaliactivity , and theMAC transduction efficiency was 4 to 20-times less than that of HIV-1NL4-3 when the two vectors were used to challenge any of a panel of T cell lines, including Jurkat, SupT1, and CEM-SS cells, the Burkitt lymphoma-derived B cell line Raji, or the myelomonocytic cell lines U937 and THP-1 . Based on these parameters, the decrease in apparent infectivity of SIVMAC did not appear to be explained by poor expression of the GFP reporter from the SIV LTR. The latter point was demonstrated more conclusively by using 3-part lentiviral vectors in which the GFP reporter was expressed from the HIV-1 and SIVMAC vectors using an identical spleen focus-forming virus (SFFV) promoter  were prepared, stimulated with PHA for three days, and challenged with the single-cycle vectors. SIVMAC transduction was less efficient than for HIV-1NL4-3 , had transduction activity more similar to that of SIVMAC239  is the retroviral determinant that confers sensitivity to several restriction factors, including Fv1 [MAC239 or HIV-2ROD. Neither of the two chimeras had transduction activity on CRFK cells or on HeLa cells.The experiments described above suggest that SIVding Fv1 , TRIM5 [ding Fv1 ,45, and ding Fv1 –57, the ROD and SIVMAC239 CA back to amino acid 202, using HIV-1 CA sequences to encode amino acids 203 to 230 . As with the 2-part vectors, no infectivity was observed unless CA amino acids 203–230 were provided by HIV-1. Among the chimeras generated, representatives from Groups AB, A, D, E, F, and H, and from a primary SIVSM, were sufficiently infectious to evaluate CRFK-normalized transduction efficiency on Jurkat T cells. As a general trend, chimeras generated with CA from the epidemic Groups (A and B) were the most infectious on Jurkat T cells, those from the non-epidemic Groups  were less infectious, and that from SIVSM was the least infectious  bearing restriction-sensitive cores [MAC-specific restriction activity in Jurkat T cells with SIV VLPs were also unsuccessful.Given the results described above, evidence was sought that the cell type-specific defect in SIVve cores . Flat, eve cores ,60. Atte2O3 rescues retroviruses from CA-specific restriction by TRIM5 but has no effect on retrovirus transduction efficiency in the absence of TRIM5-mediated restriction [2O3 blocks TRIM5-mediated restriction is not known, though the effect results in increased reverse transcription and correlates with disruption of mitochondrial membrane potential [Astriction ,47,61,62otential ,61.MAC transduction of human blood cells might be restricted by TRIM5, or by a cellular factor with similar properties, the effect of As2O3 on SIVMAC transduction was assessed. As2O3 had no effect on the transduction efficiency of VSV G-pseudotyped, 2-part vectors for either SIVMAC239 or HIV-1NL4-3 in TE671 , and the other which is not restricted (B-MLV) . The throbserved . Also, aved + T cellswo weeks .+ T cells from one of two representative blood donors are shown in + T cells, was not sufficient to assess the efficiency of TRIM5 knockdown. Instead, a lentiviral vector derived from the equine infectious anemia virus (EIAV-GFP) was utilized [NL4-3 infectivity , or the isogenic vector bearing the SIVMAC239 CA (sCA-GFP), that were shown schematically in Human blood cells such as Jurkat T cells might be less permissive for SIVA flow cytometry-based assay was established that discriminates infected heterokaryons from those cells that fail to form heterokaryons . PrimaryAs a control, HeLa-RFP cells were engineered to express TvA (HeLa-RFP-TvA); in these cells, transduction with SIV CA-GFP was 1.6-fold higher than with HIV-1 CA-GFP . ChallenMAC239 transduced human blood cells less efficiently than did HIV-1. Lower SIVMAC239 transduction efficiency relative to HIV-1 was observed with all human blood-derived cells tested here, including cell lines of lymphoid and myeloid lineage, human PBMCs and primary CD4+ T cells, and, as previously described [MAC239 as by HIV-1.The characteristics of a previously unreported retroviral restriction activity in human blood cells are described here. The first clue to the existence of this restriction activity was that SIVescribed ,50,74, mMAC239-specific, restriction factor in human blood cells—as opposed to the lack of a cofactor for SIVMAC239 replication in these cells—was supported by the finding of a block to SIVMAC239 replication in Jurkat/HeLa-heterokaryons of equal magnitude to the block in Jurkat T cells. Similar heterokaryon experiments demonstrated the presence of a dominant restriction activity prior to the cloning of the retroviral restriction factors APOBEC3G, TRIM5, and TETHERIN [The presence of a dominant-acting, SIVTETHERIN ,43,75,76TETHERIN , and theTETHERIN .MAC239-specific restriction activity described here will be called Lv4. Whether this activity is due to a single factor, or due to a multi-factor complex, remains to be determined. Knockdown experiments presented here showed that Lv4 is distinct from TRIM5 , 731744GCTCAGCAAGCAGCAGCTGACACAGGAAACAACAGCCAGGTCAGCCAAAATTACCCAGTGCAACAAGTAGCTGGCAATTATGTCCATGTGCCGTTAAGTCCCCGAACCTTAAATGCCTGGGTAAAATTAGTGGAGGAAAAGAAGTTCGGGGCAGAAATAGTACCAGGATTTCAGGCACTATCAGAGGGATGTACCCCTTATGATATCAATCAAATGCTAAATTGTGTGGGAGAACACCAGGCAGCCATGCAAGTCATTAGAGAAATAATCAATGAAGAGGCGGCAGACTGGGACCAGCAACACCCGATACCAGGTCCACTGCCAGCAGGACAACTTAGAGACCCCAGAGGATCAGATATAGCGGGAACCACCAGCACAGTAGAGGAACAAATACAGTGGATGTACAGGGGTCAAAATTCCGTCCCAGTGGGGAACATTTATAGAAGATGGATTCAATTAGGATTGCAGAAATGTGTCAGGATGTACAATCCTACTAATATACTAGATGTAAAACAAGGGCCAAAAGAACCCTTCCAAAGCTATGTAGATAGATTCTACAAAAGCCTACGGGCAGAACAAGCAGACACAGCCGTGAGAGCATGGATGACAGAAACACTACTGGTCCAGAATGCTAACCCAGATTGCAAGCTAGTACTC>HIV-2(A), GH123TGTACAACAGACAGGCGGTGGCAACTATATCCACGTGCCACTGAGCCCCCGAACTCTAAATGCTTGGGTAAAATTAGTAGAGGACAAGAAGTTCGGGGCAGAAGTAGTGCCAGGATTTCAAGCACTCTCAGAAGGCTGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTGGGCGATCACCAAGCAGCTATGCAAATAATCAGAGAGATTATCAATGACGAAGCAGCAGATTGGGATGCACAGCACCCAATACCAGGCCCCTTACCAGCAGGGCAGCTTAGAGACCCAAGGGGGTCTGACATAGCAGGAACAACTAGCACAGTAGAAGAACAGATCCAGTGGATGTATAGGCCACAAAATCCCGTGCCGGTAGGGAACATCTACAGAAGATGGATCCAGATAGGGCTACAGAAGTGTGTCAGGATGTACAACCCAACTAACATCTTAGACGTAAAGCAGGGACCAAAGGAACCGTTCCAGAGCTATGTGGACAGGTTCTATAAAAGCTTGAGGGCAGAACAAACAGATCCGGCAGTAAAGAACTGGATGACCCAAACGCTGCTAATACAGAATGCCAACCCAGACTGCAAGTTAGTACTA>HIV-2(D), L33083AGTGCAGCAAGTCGGCGGAAATTATGTCCACCTACCGCTGAGTCCCAGAACATTAAATGCATGGGTTAAGTTAGTGGAGGACAAAAAATTCGGGGCAGAGGTAGTGCCAGGGTTTCAGGCACTATCGGAAGGCTGCACTCCGTATGACATCAATCAGATGCTAAATTGTGTAGGAGAACATCAGGCAGCCATGCAGATCATAAGGGAAATAATCAATGATGAGGCAGCAGATTGGGATCAGCAGCATCCACAACCAGGCCCACTACCAGCAGGACAGCTCAGAGATCCACGAGGATCTGATATAGCAGGAACCACTAGCACAGTGGAGGAACAAATACAGTGGATGTACAGGCAGCAGAATCCCATACCAGTTGGAAATATCTATAGGAGATGGATCCAGCTAGGGTTACAGAAATGTGTCAGAATGTACAACCCAACTAACATTCTGGATATAAAACAAGGGCCAAAAGAGACGTTCCAGAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(E), L33087AGTGCAACAGATAGGAAATAACTATGTGCACTCTCCACTGTCCCCAAGAACATTGAATGCATGGGTCAAATTAGTAGAAGAAAAGAAATTTGGAGCAGAGGTAGTGCCAGGCTTCCAGGCATTATCAGAAGGATGCACCCCGTATGACATCAACCAGATGCTTAATTGCGTGGGGGAACATCAGGCAGCCATGCAAATTATCAGAGAGATAATCAATGAAGAAGCAGCAGATTGGGACGTACAGCATCCAAGAGGGCAACCGCCAGCACAGGGCCTAAGAGACCCATCAGGATCAGACATAGCAGGGACAACCAGTACCCCCGCAGAACAAATAGAGTGGATGTACAGGAATCCAAATCCAATCCCTGTGGGAGACATCTATAGAAGATGGATCCAGCTAGGGCTCCAGAAATGTGTCAGAATGTATAATCCAACAAACATTCTGGACGTCAAACAGGGGCCCAAAGAATCTTTTCAGAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(F), U75441AGTGCAGCAGGTAGGAGGAAATTACACCCATATTCCTCTGAGTCCGAGGACATTAAATGCTTGGGTTAAATTAGTAGAGGAAAAGAAATTTGGGGCAGAAATAGTGCCAGGCTTCCAAGCATTGTCAGAAGGCTGCACCCCTTATGATATTAATCAAATGTTAAATTGTGTAGGGGAACATCAGGCAGCCATGCAAATAATCAGGGAAATAATCAATGAAGAAGCAGCCGACTGGGATCAGAATCATCCAAGGCAGCTGCCAGCGCCACCAGGGCTGCGTGATCCGTCAGGATCTGACATTGCAGGAACAACTAGTACAGTACAAGAACAGATAGAATGGATGTACAGACAGGGTAACTCAATCCCAGTAGGGGACATTTACAGAAGATGGATCCAAATAGGCCTTCAAAAATGTGTAAGAATGTACAATCCTACTAATATCCTAGATGTAAAACAGGGACCAAAAGAACCATTTCAAAGCTATGTAGATAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACACAAACACTGCTGATTCAGAATGCTAACCCAGATTGCAAGTTAGTACTA>HIV-2(H), AY5308GGTGCAGCAGATAGGTGGCAATTATGCCCACCTACCTCTAAGTCCTAGAACACTCAATGCCTGGGTAAAACTGGTAGAGGAGAAAAAATTTGGAGCAGAAGTAGTGCCAGGATTTCAGGCACTCTCAGAGGGCTGCACGCCCTATGATATTAATCAAATGTTAAATTGCGTGGGAGAACATCAAGCTGCTATGCAAATTATCAGGGAAATAATTAATGATGAAGCAGCAGATTGGGACACACAGCACCCAAACCAAGGCCCACCACCAGCAGGGCAACTTAGAGAGCCAAGAGGTTCTGATATTGCAGGAACAACTAGCACAGTGGAAGAGCAGATACAGTGGATGTACAGGCCGCAAAATCCAATACCGGTGGGTAACATCTATCGGAGATGGATCCAATTGGGCCTACAAAAATGTGTTAGAATGTACAATCCAACTAATATCTTAGATATAAAGCAAGGGCCAAAGGAGCCATTTCAAAGTTATGTAGATAGATTCTACAAAAGTTTGAGAGCAGAACAAACAGATCCAGCAGTGAAAAATTGGATGACTCAGACGCTGCTGATTCAGAATGCTAACCCAGACTGCAAACTCGTGTTA>SIVSME041, HM059825AGTGCAGCAAGTAGGTGGCAATTATACCCACCTACCCTTAAGTCCAAGAACATTAAATGCTTGGGTAAAATTGATAGAAGAGAAAAAATTTGGGGCAGAAGTAGTGCCAGGATTCCAAGCACTATCAGAAGGCTGCACTCCCTATGACATCAATCAGATGCTAAATTGTGTAGGGGAGCATCAATCAGCCATGCAAATTATTAGAGAAATTATAAATGAAGAAGCTGCTGATTGGGATTTACAACACCCACAGCCAGGTCCAATACCAGCAGGACAACTTAGAGACCCGAGAGGATCAGACATTGCAGGAACTACTAGCACAGTAGAAGAACAAATTCAATGGATGTATAGGCAGCAAAACCCTATACCAGTAGGTAACATTTACAGAAGGTGGATCCAATTAGGGCTGCAAAAATGTGTAAGGATGTATAATCCAACAAACATTTTAGATGTGAAACAAGGACCAAAAGAGCCATTTCAAAGCTATGTAGATAGATTCTACAAGAGTCTAAGAGCAGAACAAACAGACCCAGCAGTGAAAAATTGGATGACTCAAACACTGCTGATTCAAAATGCTAACCCAGATTGCAAATTGGTGCTCgag-pol plasmid and pONY8.0 is an EIAV GFP-packaging vector [pMD2.G encodes the vesicular stomatitis virus glycoprotein (VSV G) and psPAX2 encodes HIV-1 Gag and Gag-Pol . pCIG3N g vector .env glycoprotein for virion pseudotyping was expressed from pAB6 [pAPM is a lentiviral vector expressing puromycin-resistance and a miR30-based knockdown cassette from the spleen focus forming virus LTR ,63,64. Trom pAB6 . Far redrom pAB6  was clonCell lines were either grown in DMEM  or RPMI , supplemented with 10% fetal calf serum as described before ,104,105.PBMC were separated by Ficoll density centrifugation, stimulated with PHA for 3 days, and cultured in RPMI supplemented with antibiotics, 10% fetal bovine serum, and 20 IU/ml hIL-2 ,106.+ T lymphocytes were enriched from PBMC by positive selection using magnetic beads (Miltenyi Biotec). Typically the resulting population was >99% CD4+. Cells were stimulated for 24 hrs on NUNC maxisorp plates that had been coated with 2 μg/ml anti-CD3 antibody and 2 μg/ml anti-CD28 antibody (BD Biosciences) in RPMI with 10% FBS, glutamax (Invitrogen), and 20 IU/ml hIL-2. Two wks after primary stimulation, cells were re-stimulated using plate-bound anti-CD3 and anti-CD28 antibodies.CD4MAC239, and the CA chimera vectors described above, were prepared by co-transfection of the indicated plasmids with pMD2.G in 293T cells, as described [6 cells were plated per 10-cm plate. The next day, cells were transfected using Lipofectamine 2000 (Invitrogen) and 20 μg of pAPM, 15 μg of psPAX2 and 5 μg of pMD2.G. Supernatant was collected and passed through a 0.45 μM filter at 48 hrs and at 72 hrs post-transfection, and used immediately to transduce target cells.VSV G-pseudotyped viral stocks of HIV-1, SIVescribed . Virion escribed  and by tescribed . For pro4 of the indicated target cells, in 0.4 ml media per well, in 24-well plates. As2O3 (Sigma) was prepared as described [Reporter virus-containing supernatant was titrated onto 4 x 10escribed  and, wheescribed .+ T cells were spinfected with shRNA-encoding APM vectors twice, at 24 hr and 48 hr after stimulation with plate-bound anti-CD3 and anti-CD28 antibodies. Spinfection was done at 1,130 rcf for 90 mins, using 2 ml of freshly produced virus supernatant for each well of a 6-well plate containing 5 x 105 stimulated lymphocytes. Cells were put in 5 μg/ml of puromycin for 72 hrs, 2 days after the first spinfection.Jurkat cells or primary CD44)2SO4, 20 mM KCl, 5 mM MgCl2, 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 μM dNTPs. All reactions and quantitation of product were carried out with a Biorad CFX96 cycler. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).Virus-containing supernatant was harvested 48 hr post-transfection, clarified by low-speed centrifugation, and filtered through 0.45 μm pore filters (Sarstedt). Reverse transcriptase (RT) activity in the supernatant was quantified using a modified Sybr green I-based, real-time PCR, enhanced RT assay ,109. VirCell-free virions were normalized by RT-activity and incubated with CRFK, Hela or Jurkat cells in 6-well plates for 12 hrs, for full-length linear cDNA and 2-LTR circles, or 48 hrs, for Alu PCR. For each virus and cell type, infections were also performed in the presence of 40 μM AZT, to control for contamination of plasmid DNA in the PCR reaction. Cells were harvested and washed extensively with PBS. Total DNA was extracted , quantified, and subjected to real-time PCR with a Biorad CFX96 cycler.4)2SO4, 20 mM KCl, 5 mM MgCl2, 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 μM dNTPs. The PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. Provirus was quantified by Taqman-based ALU-PCR according to the protocol described by Butler et al. [Full-length linear retroviral cDNA and 2-LTR circles were detected with SYBR-Green I based reactions using 100 ng template DNA and 320 nM of each primer pair  in 20 mM Tris-Cl pH 8.3, 5 mM , at 37°C. Cells were incubated for another 2 mins and then 2 ml of serum-free DMEM was added slowly over a period of 4 minutes at 37°C with constant, gentle agitation. An additional 5 ml of serum-free DMEM was added and cells were incubated for 5 min at 37°C. Cells were then pelleted and resuspended in complete medium before seeding in 24-well plates. 6 hours later, cells were challenged with ALV-A Env-pseudotyped vectors. A negative fusion control sample was also produced with no PEG addition. Infected cell cultures were analyzed using a FACS-Canto (BD) 48 hrs after vector challenge. Fluorescence acquisition was performed using blue (488 nm) and red (633 nm) lasers. Dead cells were excluded from the analysis based on propidium iodide staining.2 x 10http://grants.nih.gov/grants/policy/hs/faqs_aps_definitions.htm.Human peripheral blood mononuclear cells (PBMC) were obtained from anonymous, untraceable blood donors. This research is therefore considered non-human subjects research by our Institutional Review Board, based on NIH guidelines (45 CFR 46.102(f)):


TRHR) gene, rs1800795 in the interleukin-6 (IL6) gene and rs6552828 in the coenzyme A synthetase long-chain 1 (ACSL1) gene. To gain insight into their functionality (which is yet unknown), here we determined for the first time luciferase gene reporter activity at the muscle tissue level in rs7832552 and rs6552828. We then compared allele/genotype frequencies of the 3 abovementioned variants among centenarians  and healthy controls  of the same ethnic and geographic origin (Spain). We also studied healthy centenarians  and controls  from Italy, and centenarians  and healthy controls  from Japan. The THRH rs7832552 T-allele and ACSL1 rs6552828 A-allele up-regulated luciferase activity compared to the C and G-allele, respectively (P = 0.001). Yet we found no significant association of EL with rs7832552, rs1800795 or rs6552828 in any of the 3 cohorts. Further research is needed with larger cohorts of centenarians of different origin as well as with younger old people.There are several gene variants that are candidates to influence functional capacity in long-lived individuals. As such, their potential association with exceptional longevity  deserves analysis. Among them are rs7832552 in the thyrotropin-releasing hormone receptor ( The oldest old population (≥85 years) is rapidly expanding among westerners Waite, . HoweverMSTN) gene   gene), might also influence the likelihood of reaching EL  gene, rs16892496 and rs7832552  in the interleukin-6 (IL6) gene, where the G-allele is associated with higher transcription in vitro   analysis of 379,319 SNPs in US Caucasians of both genders (aged 50 years on average) revealed an association of lean body mass with 2 SNPs in tight linkage disequilibrium within the thyrotropin-releasing hormone receptor (2peak)], a recent GWAS study in sedentary Caucasians found that, among 324,611 SNPs, the strongest association with the VO2peak response to exercise was found to acyl coenzyme A synthetase long-chain 1 (ACSL1) gene polymorphism rs6552828 —among Spanish centenarians (cases) and healthy controls matched by ethnic and geographic origin and also in 2 other geographically and ethnically-independent replication cohorts .In order to analyze their functionality at the muscle level, we measured for the first time luciferase gene reporter activity in MluI/NheI in the 5′ and XhoI in the 3′ (see below -in bold) of the sequences obtained from the genomes of:A-allele and one individual homozygous for the rs16892496 C-allele (see below -underlined)one individual homozygous for the rs16892496 AArs16892496 AGTGGGATACAAGTCACTCTCAGGCTTGAAAAATGAGTAGGCATTCACTAGGCCAACATAAAATACAAGAAGACCCTCCAGTCTGCAGAAGTAGTCAATGACTCGAGGCTAGCTATGAAAGATCTACGTTAAAACATAAGGTTAAGCTGTGCAGTGTACAGAAGAGACAAGAAAGTGGTACTTACTGTGCATAAGGTTGAAGAGCAAGCCCCCCCrs16892496 CGTGGGATACAAGTCACTCTCAGGCTTGAAAAATGAGTAGGCATTCACTAGGCCAACATAAAATACAAGAAGACCCTCCAGTCTGCAGAAGTAGTCAATGACTCGAGGCTAGCTATGAAAGATCTACGTTAAAACATAAGGTTAAGCTGTGCAGTGTACAGAAGAGACAAGAAAGTGGTACTTACTGTGCATAAGGTTGAAGAGCAAGCCCCCC-allele and one homozygous for the rs7832552 T-allele (see below -underlined)one individual homozygous for the rs7832552 CCrs7832552-CGAATGCTCTTAATAAACAGGATCGATCAAGGGTGCTTGACTCTTGTTGTTCATGTGCAAGTATAGTGGCTTTTTTGTGCCTCAACAAAACCATCAAGAGTCTCGAGGAGCTCATTAGCCTTGTGACAAAAGCAACGCACTCCATTTTGCACACAGTACTTGACTTTATTTTGCTACTGCCTTGACCTCAAAGGAATGTGATAGTGTGAGGTATTrs7832552-TGAATGCTCTTAATAAACAGGATCGATCAAGGGTGCTTGACTCTTGTTGTTCATGTGCAAGTATAGTGGCTTTTTTGTGCCTCAACAAAACCATCAAGAGTCTCGAGGAGCTCATTAGCCTTGTGACAAAAGCAACGCACTCCATTTTGCACACAGTACTTGACTTTATTTTGCTACTGCCTTGACCTCAAAGGAATGTGATAGTGTGAGGTAA-allele and one individual homozygous for the rs6552828 G-allele (see below -underlined)one individual homozygous for the rs6552828 rs6552828-AAAAGAAAGTACAGAATAGTATTTGAGATCCTAGATGCAGCCGGACGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGTGGATCACCCTCGAGGAGCTCCAAGACATTATAGCCAAAAGAAACAAACAGATAAATTGGTGTGCATAAACTTTAAACCAACCACCAGATATCTAAAGAGGGAATACAGCACAGTGTTGGArs6552828-GGGAGAAAGTACAGAATAGTATTTGAGATCCTAGATGCAGCCGGACGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGCGGGTGGATCACCCTCGAGGAGCTCCAAGACATTATAGCCAAAAGAAACAAACAGATAAATTGGTGTGCATAAACTTTAAACCAACCACCAGATATCTAAAGAGGGAATACAGCACAGTGTTGGAThe fragment, including the allele, was directly inserted into the pGL3-promoter at the restriction recognition sites We used mice skeletal muscle C2C12 cell lines to study muscle-specific expression. We performed cell cultures, transfections and dual-luciferase reporter assays following the procedures previously reported by our group , University of Pavia , and National Institute of Health and Nutrition, (Japan) Medical Research Institute and Keio University (Japan)] and the study followed the tenets of the Declaration of Helsinki for Human Research. Written consent was also obtained from each participant.Two groups of Spanish subjects were assessed: (i) 138 cases ; and (ii) 334 healthy controls . All the subjects were of the same Caucasian (Spanish) descent for ≥3 generations. The major diseases among the centenarians were osteoarthritis (66%), hypertension (57%), dementia (51%) and cardiovascular disease . The DNA of a convenience sample of 355 younger disease-free controls with no reported family history of high longevity (>90 years) was collected during 2008-2012 in the European University of Madrid.Two groups of subjects from Northern Italy (mainly from Lombardy and Piedmont) were studied: (i) 79 cases ; and (ii) 316 healthy controls . All patients and controls were Caucasian whites of Italian descent for ≥3 generations The Italian centenarians were free of major age-related diseases, i.e., severe cognitive impairment, clinically evident cancer, CVD, renal insufficiency or severe physical impairment  descent were assessed: (i) 742 cases ; and (ii) 499 healthy controls . The group of cases was gathered from 2 cohorts, which are described in detail elsewhere  because the genotype distributions of both SNPs are completely linked according to available HapMap for both European and Asian populations (sorted as a Supplementary file 1) and previous research has shown that both SNPs are in strong linkage disequilibrium (r2 = 0.98). All genotyping was performed only for research purposes with the researchers who performed the genotyping being blinded to the participants' identities. For quality control, a random ~20% of the samples of each cohort were genotyped again, with no differences in the results compared with the initial genotyping.As mentioned above, only one DNA was extracted from the participants' buccal cells  using a standard phenol chloroform protocol and the genotype analyses were performed in the Biomedicine laboratory at the European University, Madrid (Spain). The DNA samples were diluted with sterile water and stored at −20°C until analysis. Genotyping was performed by Real-Time PCR and using the TaqMan® rs7832552, rs6552828, and rs1800795 SNP genotyping assays with a Step One Real-Time PCR System .Genomic DNA was purified from blood leukocytes using the QiaAmp DNA Mini kit  according to the manufacturer's protocol. Genotyping was performed at the Cellular Pathophysiology and Clinical Immunology Laboratory (University of Pavia) using the TaqMan® rs7832552, rs6552828, and rs1800795 SNP genotyping assays .Total genomic DNA was extracted from blood leukocytes with a QIAamp DNA Blood Mini or Maxi Kit . Genotyping of rs7832552, rs6552828, and rs1800795 was performed at the Institute of Health and Sports Science and Medicine (Juntendo University) with Real Time Thermocycler  using TaqMan SNP genotyping assay method. PCR 384-well plates were read on LightCycler 480 using the end-point analysis mode. Allelic discrimination analysis was performed with a LightCycler 480 SW software version 1.5.1.62 .2-test. Genotype/allele frequencies of cases vs. controls within each cohort  were compared using the χ2-test with Yates' correction and the association between genotypes/alleles and EL within each of the 3 cohorts was analyzed with logistic regression analysis after adjusting for sex. All statistical analyses were performed using the PASW  and corrected for multiple comparisons using the Bonferroni's method -that is, the threshold P-value was obtained by dividing 0.05 by the number of studied polymorphisms (P = 0.05/3 = 0.017).One-way analysis of variance was used to compare the relative luciferase activity in the different plasmids of each SNP. Allele frequencies were calculated by gene-counting. We tested Hardy-Weinberg equilibrium (HWE) using χP ≤ 0.001); the THRH rs16892496 A-allele up-regulated luciferase activity compared to the C-allele (upper panel), the THRH rs7832552 T-allele up-regulated luciferase activity compared to the C-allele (middle panel), and the ACSL1 rs6552828 A-allele up-regulated luciferase activity compared to the G-allele (lower panel).The results of luciferase report analyses are presented in Figure THRH rs7832552, 97.2% in cases and 100% in controls; ACSL rs6552828, 97.2% in cases and 99.1% in controls; IL6 rs1800795, 100% in cases and 94.9% in controls. The distribution of all genotypes was consistent with the HWE in both groups (P > 0.05), except for IL6 rs1800795 in the control group (P < 0.01).Rate of genotyping success was as follows: 2 = 1.21, P = 0.27) or genotype frequency distributions of THRH rs7832552 did not differ between cases and controls . Using logistic regression analysis, no significant associations were found between EL and rs7832552, including when analyzing both sexes separately (data not shown). No differences were found for IL6 rs1800795 in allele  or genotype distributions , with no significant association with EL after adjusting for sex or when analyzing both sexes separately -data not shown) or for ACSL1 rs6552828 .The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table P > 0.05), except for THRH rs7832552 in the control group (P = 0.04).Rate of genotyping success was 100% for all gene variants. The distribution of all genotypes was consistent with the HWE in both groups  and genotype frequency distributions of THRH rs7832552 did not differ between groups  and no significant association was found between this polymorphism and EL using logistic regression adjusted by sex, or when analyzing both sexes separately (data not shown). Similar results were found for IL6 rs1800795  and ACSL1 rs6552828 , with no significant association between these two polymorphisms and EL using logistic regression adjusted by sex, or when analyzing both sexes separately (data not shown).The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table THRH rs7832552, 97.0% in cases and 100% in controls; IL6 rs1800795, 98.7% in cases and 100% in controls; ACSL1 rs6552828, 95.9% in cases and 99.2% in controls. The distribution of all genotypes was consistent with the HWE in both groups (P > 0.05), except for rs6552828 in centenarians (P = 0.02).Rate of genotyping success was as follows: THRH rs7832552 , rs1800795 , or rs6552828 , with no significant association between any of the SNPs and EL using logistic regression adjusted by sex or when analyzing both sexes separately (data not shown).The results of genotype/allele frequency distributions as well as of binary logistic regression adjusted by sex are shown in Table in vitro approach) the potential functional consequences of the rs16892496, rs7832552, and rs6552828 SNPs, with the A-allele, T-allele and A-allele up-regulating luciferase activity compared to the other alleles, respectively. The THRH rs7832552 and ACSL1 rs6552828 SNPs are intronic genomic variants and, as such, could potentially alter the stability and/or alternative splicing of mRNA, as well as transcription factor binding  might favor EL. Yet a GWAS study reported that the TRHR rs7832552 SNP was associated with lean body mass in US Caucasians  genotype had 2.55 kg higher lean body mass compared to the other subjects. There is some scientific rationale in postulating that higher TRHR expression might help preservation of muscle mass in long-lived individuals: TRHR stimulates the hypothalamic-pituitary-thyroid axis, thereby leading to the release of thyroxin, a hormone that plays an important role in the development of skeletal muscle as well as in attenuating age-related changes in tissue function , we used convenience samples, which increases the risk of bias induced by population stratification. The SNPs rs1800795 and rs7832552 did not meet HWE in Spanish and Italian controls, respectively. In this regard, deviation from HWE does not necessarily reflect genotyping errors  none of them was associated with EL. Similarly, no association was found for rs1800795. More research is needed in the field with other cohorts, using larger population samples, as well as younger elderly  to assess the potential link between these genetic variants and the human aging process.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.