The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.Proximity labeling provides a powerful  Hin vivo . Proximiin vivo , 2, 7. T limited , 10 and  limited , 12 and  tissues  or pretr tissues .Caenorhabditis elegans has proven a useful workhorse to investigate metazoan biology, offering powerful genetics, in vivo cell biology, an anatomy described at electron micrograph resolution, and a defined number of cells whose gene expression can be profiled at single cell resolution  tRNA synthetase (MuPheRS)  , 27. MuPC. elegans results in robust biotinylation signals in the intestine, with the strongest signals generated by TurboID , we generated transgenic animals expressing a TurboID-mNeongreen-3xFLAG (TbID-mNG) fusion protein in various tissues (B). We compared protein biotinylation levels in age-synchronized young adults expressing the neuronal rab-3p::TbID-mNG transgene with wild-type controls. In the absence of exogenously added biotin, transgenic animals showed only a slight increase in biotinylated proteins compared with controls. Adding exogenous biotin increased protein biotinylation specifically in animals expressing the rab-3p::TbID-mNG transgene (C). To optimize biotin availability to worm tissues, we treated animals with exogenous biotin for different time intervals, using biotinylation in neurons as a readout. A 2-h incubation was sufficient to achieve robust protein labeling (D). To identify an optimum biotin concentration for protein labeling, we treated animals with varying concentrations of biotin for 2 h. We observed a substantial increase in biotinylation in worms treated with 1 mM biotin but higher biotin concentrations did not appear to further increase biotinylation (E). We also used the E. coli biotin auxotrophic strain MG1655 as a food source for worms instead of standard OP50 , indicating that TurboID is functional in all major C. elegans tissues.Like pan-neuronal expression, intestinal, hypodermal, and muscle-specific expression of TurboID-mNG conferred robust biotinylation activity A, indicaC. elegans (A–C). A significant advantage of PL compared with IP is that extracts can be collected under strong denaturing conditions , since the biotin tag is covalently attached. This allowed us to achieve >95 % solubilization of proteins . After extensive washing and elution, we fractionated the affinity-purified biotinylated proteins by SDS-PAGE, visualized the proteins by Coomassie staining, and analyzed gel slices by LC-MS/MS analysis (B). Using a threshold of at least two unique peptides, we cumulatively identified >4000 proteins expressed in one or more tissues . In addition, we observed a strong correlation between the two replicates at the level of spectral counts (B). Consistent with previous findings, we identified most proteins in the intestine, followed by the hypodermis and muscle cells (B), (B), and 1274 proteins were detected in only one tissue (C). Proteins identified by MS/MS when TurboID was targeted to neurons included broadly expressed neuronal proteins, such as CLA-1, UNC-31, and proteins expressed in subsets of neurons, such as the glutamate decarboxylase UNC-25, which is known to be expressed in 26 neurons, and OSM-10, which is expressed in four pairs of neurons (D). Muscle-specific samples were enriched in CPNA-1, CPNA-2, PQN-22, and F21C10.7 (D); intestine-specific samples were enriched in ACOX-2, CGR-1, C49A9.9, and C49A9.3 (D), and hypodermis-specific samples included F17A9.5, PAH-1, R05H10.1, and CPN-2 (D). In addition, we identified tissue-specific enrichment for many proteins whose expression was previously uncharacterized . For some of these proteins we validated the tissue-selective expression highlighted by our MS/MS data by making transgenic reporters. The reporters confirmed the expression profile predicted by MS/MS, validating the method's specificity .We next optimized a protocol to extract and affinity purify biotinylated proteins from  elegans , A–C. A ins A–C, . We achiins A–C, . Westernins A–C, A. After analysis B. Using B and C, . Using tB and C, , we lookB and C, A. In addl counts B. Consisle cells B, 12). . C. eleglls (B), B, and 12e tissue C. Protei neurons D. MuscleF21C10.7 D; intest C49A9.3 D, and hynd CPN-2 D. In addcterized , Fig. 2EC. elegans nervous system includes 118 classes of neurons, with most classes consisting of a single pair of neurons that form left/right homologs (gcy-8 promoter (A). Western blot analysis of extracts from these animals revealed a biotinylation signal significantly higher than that in extracts from wild-type controls (B). Correlation plots of mass spectrometry data obtained for affinity-purified extracts made from animals expressing the gcy-8p::TbID-mNG transgene showed reproducible results between replicates (A). As expected, these samples were enriched for proteins specifically or selectively expressed in AFD neurons when compared with similarly processed extracts from nontransgenic controls, or from animals expressing a rab-3p::TbID-mNG transgene (C and S4B). Enriched proteins included the transmembrane guanylate cyclases GCY-8 and GCY-18 and the homeobox transcription factor TTX-1 (D and S4B). Our MS data also identified other proteins enriched in the AFD-specific TurboID samples compared with the pan-neuronal TurboID controls (E and S4C). To examine if these proteins were selectively expressed in AFD neurons, we generated transgenic reporter lines (F). nex-4 and F37A4.6 were expressed specifically in AFD, albeit F37A4.6 at low levels; T06G6.3 was expressed in a small subset of neurons that included AFD (F). Our list of AFD-enriched proteins, identified by mass spectrometry, was consistent with AFD-specific gene expression identified by RNA Seq . ELKS-1 is expressed throughout the nervous system and localizes to the presynaptic active zone, in proximity to other presynaptic proteins such as α-liprin and RIM . Western blot analysis of extracts from ELKS-1::TbID animals did not show an increased biotinylation signal compared with wild-type controls (B). However, mass spectrometry analysis of streptavidin-purified proteins from this knock-in strain revealed enrichment of known synaptic proteins including UNC-10/RIM, SYD-1/SYDE1, SYD-2/α-liprin, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, and the RIM-binding protein RIMB-1 (D). We measured enrichment by comparing mass spectrometry data obtained for control extracts processed in parallel from wild-type and from transgenic animals expressing free TurboID-mNG throughout the nervous system, rab-3p::TbID-mNG (C and S4E) or both nervous system and other tissues (G). Between-replicate correlation plots revealed reproducible results between experimental repeats (D). We also identified several previously uncharacterized proteins as enriched in ELKS-1::TbID-mNG samples . We expressed mNeongreen translational fusion transgenes for several of these proteins exclusively in the AFD neuron pair and showed that they colocalized with ELKS-1::mScarlet at presynaptic active zones where AFD is known to synapse with AIY interneurons . Together, our findings show that TurboID-mediated proximity labeling is an effective method to reveal protein interactors in C. elegans at endogenous levels with specificity and sensitivity.We next asked if TurboID can highlight the interactome of a specific  and RIM . To havesynapses A. Westercontrols B. Howeven RIMB-1 D. We meaTbID-mNG C and S4E tissues G. Betwee repeats D. We alsrneurons  . Human ANKS1B, also known as AIDA-1, is a risk locus for autism and neurodevelopmental defects (C. elegans ortholog of human KIAA0930. C16E9.2 was recently given the name sentryn (STRN-1). STRN-1 together with the SAD kinase and liprin-α promote dense-core vesicle (DCV) pausing at presynaptic regions  also suggest that these proteins are expressed in neurons. Such hypotheses raised by the MS data need to be directly tested, for example, using colocalization studies with fluorescently tagged reporters.Some proteins identified in the ELKS-1 proximity labeling experiments are usually considered nonneuronal. We speculate some of these proteins may also be expressed in neurons. For example, we find components of the muscle dense body, including the myotilin ortholog KETN-1/MYOT, the ALP-Enigma protein ALP-1, the sorbin homolog SORB-1, and ZYXin ZYX-1  enrichedC. elegans, adding biotin to plates seeded with bacteria (E. coli OP50 or NA22 strains) is sufficient to promote TurboID labeling, but long incubation times are required to achieve detectable protein biotinylation . We find that a 2-h incubation with exogenous biotin is sufficient for strong TurboID signals in worm neurons (D). Since in the lab C. elegans is typically grown on E. coli as a food source, feeding biotin-auxotrophic E. coli to worms provides some control over the start of biotinylation by TurboID. For time-sensitive experiments, it may be possible to soak worms in a buffer containing biotin for shorter periods, e.g., 30 or 60 min and still achieve robust protein biotinylation, although further analysis using MS is required to confirm this.An important variable for TurboID protocols is the delivery of biotin or biotin derivatives. In cultured mammalian cells expressing TurboID or miniTurbo, adding biotin for as little as 10 min yields robust biotinylation . In C. e neurons D. Since C. elegans proteome comprises integral membrane proteins; however, only 10–11% of the proteins for which we detect peptides correspond to integral membrane proteins. Membrane protein identification is a long-standing problem in mass spectrometry: integral membrane proteins typically yield fewer tryptic peptides, due to a paucity of positively charged residues. In TurboID experiments underrepresentation is compounded, since biotinylation occurs on solvent exposed lysine residues. It will be interesting in future work to explore the recovery of membrane proteins when TurboID is targeted to plasma membrane of C. elegans cells.About 27% of the A) and MS analysis (data not shown). These carboxylases; PCCA-1/PCCA , PYC-1/PC (pyruvate carboxylase), MCCC-1/MCCC (methylcronotoyl coenzyme A carboxylase), POD-2/ACACA (acetyl coenzyme A carboxylase) were the most abundantly detected proteins in our MS experiments in every condition tested, including controls. Depending on how broadly and strongly we expressed TurboID in C. elegans tissues, these carboxylases account for 8%–85% of peptides identified in MS analysis. We aim to knock-in tags into each of these four genes , using CRISPR/Cas9 to allow their depletion by affinity purification, thereby increasing the sensitivity of proximity labeling, particularly when the TurboID-tagged protein is expressed in a small number of cells or at low levels.Four endogenously biotinylated carboxylases dominate the biotinylated proteome in TurboID expressing lines, as seen by both Western blot A and MS C. elegans community to map the proteomes of specific cells and subcellular structures and to characterize the interactomes of proteins of interest.Despite extensive washing and use of denaturing conditions, streptavidin purified samples retain substantial nonspecific background. Notably, the mass spectrometer identifies many proteins even when we apply our affinity purification protocol to extracts made from control animals that do not express TurboID. These nonspecific contaminant proteins may be sticky and/or expressed at high enough levels to come through to our pipeline. Alternatively, there may be endogenous biotinylation of many proteins beyond the highly abundant carboxylases PCCA-1, PYC-1, POD-2, and MCCC-1. A future priority is to create a comprehensive list of such contaminants. In summary, our findings show that TurboID works well in a wide array of contexts in the worm. TurboID will be a reliable and useful tool for the E. coli MG1655. C. elegans husbandry otherwise followed standard laboratory culture conditions (Worms were grown at room temperature (22 °C) on nematode growth medium (NGM) plates seeded with the biotin auxotroph nditions .C. elegans strains used in this study include:N2, the wild-type Bristol straindbIs37[rab-3p::mNeongreen::3XFLAG]AX7647 dbIs24[rab-3p::CeTurboID::mNeongreen::3XFLAG]AX7526 dbIs25[ges-1p::CeTurboID::mNeongreen::3XFLAG]AX7542 dbIs28[myo-3p::CeTurboID::mNeongreen::3XFLAG]AX7578 dbIs33[dpy-7p::CeTurboID::mNeongreen::3XFLAG]AX7623 AX7606 dbIs32[gcy-8p::CeTurboID::mNeongreen::3XFLAG]dbEx1234AX7917 dbEx1241AX7922 AX7928 dbEx1247AX8059 dbEx1266[R10E8.8p::mNeongreen::3XFLAG]AX7935 dbEx1251; dbEx1253AX7937 dbEx1255; dbEx1253dbEx1256[T06G6.3p::mNeongreen::3XFLAG]; dbEx1253AX7939 dbEx1296AX8110 dbEx1297AX8111 dbEx1300AX8117 elks-1(syb1710). The syb1710 allele is a knock-in that expresses ELKS-1 tagged C-terminally with CeTurboID::mNeongreen::3XFLAG.PHX1710 C. elegans  pEntry vector, which was a gift from Dr Dominique Glauser (University of Fribourg).DNA encoding N linker::CeTurboID::c-Myc and mNeongreen::3XFLAG were stitched together by fusion PCR using primer sequences that encoded a short Gly-Ser linker. The resulting PCR product (N linker::CeTurboID::c-Myc::mNeongreen::3XFLAG) was cloned into position 2 of a Multisite Gateway pDONR vector (ThermoFisher).https://www.addgene.org/.The codon-optimized TurboID plasmid is available at Addgene C. elegans genomic DNA by PCR and cloned into position 1 of a Multisite Gateway Donor vector (Thermo Fisher). The resulting pEntryslot1 vectors containing these promoters were each mixed with the dg398 pEntryslot2_mNeongreen::3XFLAG::stop vector, a pEntryslot3 vector containing the let-858 or tbb-2 3′UTR and pDEST (ThermoFisher) in an LR reaction. The resulting expression vectors were injected into the gonad of day 1 adult N2 worms at a concentration of 25 ng/uL.Promoters for B0379.1 (∼0.9 kbp), T01G5.1 (∼2.1 kbp), Y53G8AL.1 (∼2.5 kbp), nex-4 (∼1.6 kbp), F37A4.6 (∼2.5 kbp), T06G6.3 (∼2.2 kbp), and R10E8.8 (∼1.6 kbp) were amplified from C03H5.6 (∼2 kbp) and C11E4.6 (∼6 kbp) were amplified from C. elegans genomic DNA by PCR and cloned into a position 2 Gateway donor vector. cDNA for H06I04.1 isoform a was synthesized by IDT  and cloned into a position 2 Gateway donor vector. The resulting pEntryslot2 vectors were mixed with dg397 pEntryslot3_mNeongreen::3XFLAG::stop::unc-54 3′UTR entry vector and a pDEST vector in an LR reaction. elks-1 cDNA (∼2.5 kbp) was PCR-amplified from a C. elegans cDNA library and cloned into a position 2 Gateway donor vector, mixed with gcy-8 promoter (position 1) and mScarlet (position 3) and a pDEST vector in an LR reaction. Resulting expression vectors were injected into the gonad of day 1 adult N2 worms at a concentration of 25 ng/μL.ORFs for Around 100 L4 stage transgenic animals expressing an extrachromosomal array were picked and transferred to unseeded NGM plates. The plates were placed in a UV cross-linker  with lids removed and exposed to UV (energy setting 300). The irradiated worms were then transferred to seeded plates (10 worms/plate). In total, 200 of their F1 progeny expressing the transgene were picked and individually transferred to a seeded plate. Plates containing many transgenic F2 progeny, indicating potential integration of the transgene, were selected, and eight F2 animals from the candidate plates singled and examined for 100% transmission in the next generation. Integrants were outcrossed to N2 worms at least four times.F-myo-3p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGTGTAGGCAATCAGTCAAACCGAATAAAAR-myo-3p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTCTAGATGGATCTAGTGGTCGTGGGTTF-dpy-7p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGAATCTCATTCCACGATTTCTCGCAACACATR-dpy-7p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTATCTGGAACAAAATGTAAGAATATTCTTAF-gcy-8p-Entry1-GGGGACAACTTTGTATAGAAAAGTTGGGTTCAACAAGGGTATTGTATTGCAATCAGTGR-gcy-8p-Entry1-GGGGACTGCTTTTTTGTACAAACTTGTTTGATGTGGAAAAGGTAGAATCGAAAATCCF-T01G5.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCACCTGAACACAACATTTTCTGR-T01G5.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGGACATGAAATTGTATCTGAAAGCF-Y53G8AL.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGTAGTTATGGAAAAGCAACGTCGGAGR-Y53G8AL.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGTCTAAAATAGCATTGGTTCTGAAACTTTGF-B0379.1p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGAAACAATATTATTTTTGTTTCACAGR-B0379.1p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGGTTGTTGTTGTCTCGATGGAAAAGF-nex-4p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCCTGAGAATTACTGAAGTTTAAGCR-nex-4p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCTCGAGTTACTTCAATGCTCACGF-F37A4.6p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGGATTCAGAAAATTCAGAAAGGCATTCR-F37A4.6p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCATTATAGGAAGACTGAGATTCCAAGCF-T06G6.3p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCATTTTTTGCTCTAAAGGTGGAATAGR-T06G6.3p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGCTTTGAAAAAAGTTCAGAGTAGTAGAGF-C03H5.6-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGTTAGAATGTATACATCCAACATR-C03H5.6-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTATGTACGATTCATCAAACCACCTACF-C11E4.6-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGAGCCTCAAAGACTTTGTCATATCR-C11E4.6-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAATTTCTTTGGTTCTCAGTAGTTTGCTGF-H06I04.1-Entry2- GGGGACAAGTTTGTACAAAAAAGCAGGCTtttcagaaaaATGAGCAAAGAAACTGGAAAAATGGCGGR- H06I04.1-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAAAATGGTCCAGATCTTGCTTCATTGGTCF-R10E8.8p-Entry1- GGGGACAACTTTGTATAGAAAAGTTGCACATAAAATACGTTTTAGTAGCTGTCAGCACR-R10E8.8p-Entry1- GGGGACTGCTTTTTTGTACAAACTTGTTTTTGTCTGAAAATCGAACATTAAAAATAACAGGF-elks-1-cDNA-Entry2-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTTCAGAAAAATGGCACCTGGTCCCGCACCATACAGCR-elks-1-cDNA-Entry2- GGGGACCACTTTGTACAAGAAAGCTGGGTAGGCCCAAATTCCGTCAGCATCGTCGTGrab-3 promoter (∼1.2 kb) and ges-1 promoter (∼3.4 kb) were used from de Bono lab plasmid collection.N/C terminus Gly-Ser rich linkersCeTurboIDIntronsC-mycmNeongreen3XFLAGGGAGGTGGTGGATCAGGCTCGGGAGGTCGAGGCTCAGGATCCGGTTCCGGCTCCGGCTCTGGTTCCGGTTCGGGTTCCGGTTCTGGAAAGGATAACACCGTTCCACTTAAGCTTATCGCCCTTCTTGCCAACGGAGAATTCCACTCTGGAGAGCAACTTGGAGAGACTCTTGGAATGTCCCGTGCTGCCATCAACAAGCATATCCAAACCCTTCGTGATTGGGGAGTTGATGTTTTCACTGTTCCAGGAAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGATACTCCCTTCCAGAGCCAATCCCACTTCTTAACGCCAAGCAAATCCTTGGACAACTTGATGGAGGATCCGTCGCTGTCCTTCCAGTTGTTGATTCCACCAACCAATACCTTCTTGACCGTATCGGAGAGCTTAAGTCTGGAGACGCCTGCATCGCTGAGTACCAACAAGCTGGACGCGGATCTCGCGGACGCAAGTGGTTCTCCCCATTCGGAGCCAACCTTTACCTTTCTATGTTCTGGCGTCTTAAGCGTGGACCAGCTGCTATCGGACTTGGACCAGTTATCGGAATCGTTATGGCTGAGGCCCTTCGTAAGCTTGGATATAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTTCGTGTTAAGTGGCCAAACGATCTTTACCTTCAAGACCGTAAGCTTGCTGGAATCCTTGTCGAGCTTGCTGGAATCACCGGAGACGCCGCTCAAATCGTTATCGGAGCTGGAATCAACGTTGCCATGCGTCGTGTTGAGGAGTCTGTTGTTAACCAAGGATGGATCACTCTTCAAGAGGCTGGAATCAACCTTGATCGTAACACCCTTGCTGCCACCCTTATCCGTGAGCTTCGTGCTGCCCTTGAGCTTTTCGAGCAAGAGGGACTTGCCCCATACCTTCCACGCTGGGAGAAGCTTGACAACTTCATCAACCGCCCAGTTAAGCTTATCATCGGAGATAAGGAATCTTCGGAATCTCTCGCGGAATCGACAAGCAAGGAGCTCTTCTTCTTGAGCAAGATGGAGTCATTAAGCCATGGATGGGAGGAGAGATTTCCCTTCGTTCCGCTGAGAAGGCCGGAGGAGAACAGAAGCTTATAAGTGAGGAGGACCTGGGATCCGCTGGATCCGCTGCTGGATCCGGTGAGTTCATGGTGTCGAAGGGAGAAGAGGATAACATGGCTTCACTCCCAGCTACACACGAACTCCACATCTTCGGATCGATCAACGGAGTGGATTTCGATATGGTCGGACAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGAACTGGAAACCCAAACGATGGATACGAGGAACTCAACCTCAAGTCGACAAAGGGAGATGCAATTCTCGCCATGGATTCTCGTGCCACACATCGGATACGGATTCCACCAATACCTCCCATACCCAGgtaagtttaaactgagttctactaactaacgagtaatatttaaattttcagATGGAATGTCACCATTCCAAGCTGCCATGGTGGATGGATCGGGATACCAAGTTCACCGAACAATGCAATTCGAGGATGGAGCCTCGCTCAGTGAACTACCGATACACATACGAGGGATCGCACATCAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGGAGAGGCTCAAGTTAAGGGAACAGGATTCCCAGCTGATGGACCAGTGATGACAAACTCACTCACAGCTGCTGATTGGTGCCGATCGAAAAAGACATACCCAAATGATAAGACAATATCTCGACATTCAAGTGGTCGTACACTACTGGAAACGGAAAGCGATACCGATCGACAGCCCGAACAACATACACATTCGCTAAGCCAATGGCCGCCAACTACCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAATCAACCAATGTACGTGTTCCGAAAGACAGAACTCAAGCACTCAAAGACAGAGCTGAACTTCAAAGAGTGGCAAAAGGCCTTCACAGATGTGATGGGAATGGATGAACTCTACAAGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGC. elegans expressing fluorescent proteins were acquired using a Leica  SP8 inverted laser scanning confocal microscope with 10× 0.3 NA dry, 63× 1.2 NA water or 63× 1.2 NA oil-immersion objectives, using the LAS X software platform (Leica). The Z-project function in Image J  was used to obtain the figures used in the panels. Animals were mounted on 2% agarose pads and immobilized with 100 μM of sodium azide.Confocal microscopy images of transgenic C. elegans grown on E. coli MG1655 were harvested at L4 or young adult stage, washed three times in M9 buffer, incubated at room temperature (22 °C) in M9 buffer supplemented with 1 mM biotin, and E. coli MG1655 for 2 h. Two hours later, the worms were washed three times in M9 buffer and flash frozen after the M9 buffer was completely aspirated and 4× Bolt LDS sample buffer supplemented with fresh DTT. The samples were then thawed, boiled for 10 min at 90 °C, vortexed for 10 min, centrifuged for 30 min at 15,000 rpm at 4 °C, and the supernatant collected. Proteins were transferred to a PVDF membrane (Thermofisher Scientific) following electrophoresis using Bolt 4–12% Bis-Tris Plus gels (Thermofisher Scientific). Membranes were blocked for 1 h at room temperature with TBS-T buffer containing 5% BSA and incubated for 2 h at room temperature with HRP-conjugated or fluorescently labeled streptavidin or with HRP-conjugated antibodies. Membranes were then washed three times with TBS-T. The following antibodies or protein-HRP conjugates were used for this study: IRDye 800CW Streptavidin (1:7000 in TBS-T) (LI-COR Biosciences), Anti-FLAG M2-Peroxidase (1:5000 in TBS-T) (A8592 Sigma), anti-alpha tubulin-HRP  (DM1A Abcam ab40742), Streptavidin-HRP (1:5000 in TBS-T) . Membranes were imaged using ChemiDoc the Imaging System .Synchronized populations of C. elegans were bleached and the eggs transferred to NGM plates seeded with E. coli MG1655 to obtain synchronized populations of worms. The animals were harvested at L4 or young adult stage, washed three times in M9 buffer, incubated at room temperature (22 °C) in M9 buffer supplemented with 1 mM biotin, and E. coli MG1655 for 2 h unless stated otherwise. Two hours later, the worms were washed three times in M9 buffer and allowed to settle on ice after the last wash. After completely aspirating the M9 buffer, two volumes of RIPA buffer supplemented with 1 mM PMSF and cOmplete EDTA-free protease inhibitor cocktail (Roche Applied Science) were added to one volume of packed worms. The animals were again allowed to settle on ice and then added dropwise to liquid N2 to obtain frozen worm “popcorn.” A Spex 6875D cryogenic mill was used to grind frozen C. elegans to a fine powder, which was then stored at –80 °C. Worm powder was thawed by distributing it evenly along the length of a 50 ml falcon tube and rolling it on a tube roller at 4 °C. After the sample was completely thawed, it was centrifuged  to collect the sample at the bottom of the tube. SDS and DTT were added to the sample to final concentrations of 1% and 10 mM respectively, from stock solutions of 20% SDS and 1M DTT. The tubes were gently inverted a few times and immediately incubated at 90 °C for 5 min. After heat treatment, the samples were sonicated continuously for 1 min twice, with brief cooling between the two sonication steps. Sonication used a probe sonicator microtip (MSE Soniprep 150 plus) and an amplitude setting of 16/max. The samples were cooled to room temperature following sonication and adjusted to 2 M urea using a stock solution . The samples were then centrifuged at 100,000g for 45 min at 22 °C, and the clear supernatant between the pellet and surface lipid layer transferred to a new tube.Gravid adult g for 5 min (or until the buffer was completely eluted from resin). Around 4 ml of clarified sample was then loaded onto the equilibrated spin column and desalted by centrifugation at 1000g for 5 min (or until the sample was completely eluted from resin) to remove free biotin. The desalting step was repeated once more using freshly equilibrated columns. Protein concentration in the samples was measured using Pierce 660 nm protein assay reagent supplemented with Ionic Detergent Compatibility Reagent (IDCR) (Thermo Fisher Scientific).Zeba spin desalting columns (7K MWCO) (Thermofisher) were equilibrated three times with 5 ml RIPA buffer containing 1% SDS and 2 M urea, freshly supplemented with protease inhibitors  by centrifugation at 1000C. elegans total protein lysate) were equilibrated in RIPA buffer by briefly incubating them three times in the buffer and using magnetic separation to retain beads while discarding buffer (note: we were able to reduce the amount of protein lysate to 5–10 mg in subsequent experiments without compromising the mass spectrometry data). Desalted, clarified samples were combined with Dynabeads in a 50 ml falcon tube and incubated overnight in a tube roller at room temperature. Beads were magnetically separated using a neodymium magnet taped to the tube and incubated on a rocking platform. Unbound lysate was removed and the Dynabeads transferred to 2 ml LoBind protein tubes (Eppendorf). Beads were washed five times with 2% SDS wash buffer  and five times with 1M KCl wash buffer; beads were transferred to new tubes after each wash.Dynabeads MyOne streptavidin C1  to saturation in sample buffer (NuPAGE LDS sample buffer 4×) containing reducing agent (NuPAGE sample reducing agent 10×). Elution sample buffer was centrifuged at 13,000 rpm for 5 min to remove undissolved biotin and the elution sample buffer saturated with dissolved biotin was transferred to a new tube. Hundred microliter of elution buffer was applied to Dynabeads, vortexed gently, heated for 5 min at 90 °C, before the vortexing and heating steps were repeated. Dynabeads were separated magnetically and elution buffer containing biotinylated proteins was transferred to a new 1.5 ml LoBind protein tube (Eppendorf). Seventy microliter of sample was electrophoresed on a NuPAGE 4–12% Bis-Tris protein gel (Invitrogen), which was then stained with InstantBlue (Expedeon) for visualization. The gel was sliced into 20 fractions and sent for mass spectrometry analysis.via a nano-flow electrospray ionization source with a hybrid quadrupole orbitrap mass spectrometer . Data collection was performed in data-dependent acquisition (DDA) mode with an r = 70,000 (@ m/z 200) full MS scan from m/z 380–1600 with a target AGC value of 1e6 ions followed by 15 MS/MS scans at r = 17,500 (@ m/z 200) at a target AGC value of 1e5 ions. MS/MS scans were collected using a threshold energy of 27 for higher energy collisional dissociation (HCD) and a 30 s dynamic exclusion was employed to increase depth of coverage. Acquired raw files were then searched in MaxQuant (C. elegans reference proteome from UniProt KB (including SwissProt and TrEMBL entries) downloaded on September 27, 2019. In total, 28,474 accessions were actually searched. We used trypsin, which cleaves peptides at the C-terminal side of lysine and arginine residues, to generate peptide fragments for mass spectrometry. Most parameters were kept at their default value. Carbamidomethyl (C) (+57.0214637236) was set as fixed modification. Variable modifications included were “Oxidation (M)” (+15.9949146221), “Acetyl (Protein N-term)” (+42.0105646863), “Deamidation (NQ)” (+0.9840155848), “Gln->pyro-Glu” (-17.0265491015), “Phospho (STY)” (+79.9663304084), as well as two custom modifications  to account for TurboID-induced biotinylated peptides. Match Between Runs  and Second Peptide Search were activated. All FDRs were set to 1%. Identified data was then reprocessed in R using the evidence.txt results table, but a decision was made to exclude indirect, MBR-based identifications to focus on higher confidence hits. The decoy used to establish the FDR was the reverted search database. MaxQuant applies a PSM-level FDR globally as well as per class of modified peptides ("Site decoy fraction), and also at protein level. All three FDR levels were set to 1%.Polyacrylamide gel slices (1–2 mm) containing the purified proteins were prepared for mass spectrometric analysis using the Janus liquid handling system . Briefly, the excised protein gel pieces were placed in a well of a 96-well microtitre plate, destained with 50% v/v acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated with 55 mM iodoacetamide. After alkylation, proteins were digested with 6 ng/μl Trypsin  overnight at 37 °C. The resulting peptides were extracted in 2% v/v formic acid, 2% v/v acetonitrile. The digest was analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC (ThermoScientific Dionex) to deliver a flow of approximately 300 nl/min. A C18 Acclaim PepMap100 5 μm, 100 μm × 20 mm nanoViper (ThermoScientific Dionex), trapped the peptides prior to separation on an EASY-Spray PepMap RSLC 2 μm, 100 Å, 75 μm × 250 mm nanoViper column (ThermoScientific Dionex). Peptides were eluted using a 60-min gradient of acetonitrile (2% to 80%). The analytical column outlet was directly interfaced MaxQuant  (versionC. elegans, for which there is no requirement for review and approval from an institutional animal care and use committee. Transgenic experiments were carried out following IST guidelines for such work.The work used the free-living nematode via the PRIDE partner repository at http://www.ebi.ac.uk/pride with the dataset identifier PXD027068. Submission details: Project Name: Interactome analysis of C. elegans synapses by TurboID-based proximity labeling. Project accession: PXD027068. We have uploaded the mass-labeled MS/MS data on MS Viewer, available at https://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer. The key to access the repository is bbfl7oetve. Only ∼100,000 of the ∼2.5 million spectra obtained in the course of our work are in the database, since replicates are omitted, and the best spectrum retained for each peptide. All remaining data are contained within the article.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium This article contains The authors declare that they have no conflicts of interest with the contents of this article.


In the article titled “Long Noncoding RNA TUG1/miR-29c Axis Affects Cell Proliferation, Invasion, and Migration in Human Pancreatic Cancer” [Correct forward sequence should read as follows:5′-gatccGCTTGGCTTCTATTCTGAATCCTTTCAAGAGAAGGATTCAGAATAGAAGCCAAGCTTTTTTG-3′.Correct reverse sequence should read as follows:5′-CAAAAAAGCTTGGCTTCTATTCTGAATCCTTCTCTTGAAAGGATTCAGAATAGAAGCCAAGCG-3′.


KO-heart) die by 9 weeks of age due to extensive cardiomyocyte apoptosis and severe HF, which suggests that fortilin sustains cardiomyocyte viability. The lack of fortilin is also associated with drastic upregulation of p53 target genes in the hearts. The heart-specific deletion of p53 in fortilinKO-heart mice extends their life spans from 9 to 18 weeks by mitigating cardiomyocyte apoptosis. Our data suggest that fortilin is a novel cardiac p53 inhibitor and that its inadequate expression in failing hearts and subsequent overactivation of the p53 apoptosis pathway in cardiomyocytes exacerbates HF.Heart failure (HF) has reached epidemic proportions in developed countries, affecting over 20 million people worldwide. Despite modern medical and device therapies, 60–70% of HF patients still die within 5 years of diagnosis as it relentlessly progresses through pervasive apoptotic loss of cardiomyocytes. Although fortilin, a 172-amino-acid anti-p53 molecule, is one of the most expressed proteins in the heart, its precise role there has remained unknown. Also unclear is how cardiomyocytes are protected against apoptosis. Here, we report that failing human hearts express less fortilin than do non-failing hearts. We also found that mice lacking fortilin in the heart (fortilin Heart failure (HF)—a complex clinical syndrome secondary to structural and functional impairments of the heart muscle—has reached epidemic proportions in developed countries, currently affecting over 20 million people worldwide. Despite modern medical and device therapies, 30–40% of HF patients die within 1 year and 60–70% die within 5 years of diagnosis [Fortilin  is a highly conserved, 172-amino-acid, 20 kDa protein that blocks apoptosis –10, and To evaluate the role of fortilin in the heart and in human HF, we subjected tissue lysates of human hearts from subjects with non-failing hearts (NFHs) and HF patients with non-ischemic cardiomyopathy (NICM) and ischemic cardiomyopathy (ICM) from the Duke Human Heart Repository  to an auflox/flox mice using standard homologous recombination techniques [fortilin KO mice  by crossing fortilinflox/flox mice with mice over-expressing Cre-recombinase under the control of the cardiac-specific Myh6 promoter [+/+, Jackson Laboratory)  mice as the control. As expected, fortilin was not detectable in the hearts of fortilinKO-heart mice, whereas it was normally expressed in all other tissues at both message  Fig. . In cardces Fig.  is excisces Fig. , while fces Fig. . We usedage Fig.  and protage Fig.  levels.KO-heart mice started to die as early as 6 weeks of age and all were dead by 9 weeks of age  in fortilinKO-heart mice, but not in fortilinWT-heart mice  than in fortilinKOp53WT and fortilinKOp53KO mice (the latter two had comparable expression), both at the message  and protein  levels. In addition, total p53 expression levels were significantly higher in fortilinKOp53WT than in fortilinKOp53KO mice, both at the message  and protein levels . p53 expression levels also were significantly higher in fortilinKOp53WT than in fortilinWTp53WT mice, both at the message  and protein levels . On the other hand, phosphorylated p53, an active form of p53, was most abundant in the hearts of fortilinKOp53WT mice, followed by those of fortilinWTp53WT and then by those of fortilinKOp53KO , suggesting that the lack of fortilin increases not only total p53 protein but also phosphorylated and activated p53 protein.To test the hypothesis that the lack of fortilin causes the heart to fail through overactivation of the p53 apoptotic pathway, we knocked out p53 by crossing fortilinlox mice  and geneice Fig.  and S2b.protein levels of total p53 to be significantly higher in the heart of fortilinKOp53WT mice than in those of fortilinWTp53WT mice as we observed above. However, this process does not explain why the message levels of p53 were also significantly higher in the hearts of fortilinKOp53WT mice than in those of fortilinWTp53WT mice .Because fortilin binds p53 and facilitates its proteasome-mediated degradation at the protein level , it is ep53-pomoter-GLuc/SEAP cells). We then transiently silenced fortilin by treating the cells with lentiviral vector containing shRNA against fortilin (sh-fortilin). These cells were irradiated with a moderate X-ray dose (8 Gy) and subjected to the GLuc-SEAP assay  and the secreted alkaline phosphatase (SEAP) gene under the control of the constitutional CMV promoter . Echocardiography showed that the hearts of fortilinKOp53KO mice had better overall heart function . These data suggest that fortilin sustains heart function by negatively regulating p53 and that the lack of fortilin leads to inappropriate overactivation of the p53 pathway, apoptosis and loss of cardiomyocytes, dilated cardiomyopathy, HF, and death.Immunohistochemical staining of myocardium showed that the lack of p53 drastically reduced the expression levels of both BAX and cleaved lamin, which are apoptosis markers Fig. . ConsistKO-heart mice increased their survival by about 10 weeks .Although the deletion of p53 in the heart of fortilineks Fig. , it did ice Fig. . We founway Fig. , a vs. cKO-heart mice, we treated fortilinKOp53KO mice with either KIRA6, a selective IRE1α inhibitor [KOp53KO mice to live modestly longer compared to vehicle treatment . At week 10 of treatment (about 18 weeks of age), both body weights and HW/BW ratios of KIRA6-treated fortilinKOp53KO mice were modestly but significantly greater than those of vehicle-treated fortilinKOp53KO mice  level and by activation of the p53 promoter by knockdown of fortilin in the cardiomyocyte cell line . Because there are more endothelial cells and fibroblasts combined than cardiomyocytes in the murine hearts [KOp53KO heart. To evaluate the status of activated and functional p53 in the heart, we stained the tissue with anti-phospho-p53 antibody. We found that phosphorylated p53 were significantly less in fortilinKOp53KO mice than that in fortilinWTp53WT mice, suggesting that despite the similar total p53 signals between fortilinKOp53KO and fortilinWTp53WT hearts by both immunohistochemistry  and Western blots , functional p53 is significantly less in fortilinKOp53KO hearts than in fortilinWTp53WT hearts.There were p53 signals detectable in the Western blot of the total heart lysates from fortiline hearts –32, it iBecause cardiomyocytes are not capable of proliferating in normal conditions, their continuous apoptotic loss leads to irreversible progression of HF. Protecting cardiomyocytes against apoptosis represents a new approach in HF gene therapy, distinct from the ones focusing on calcium metabolism and β-adrenergic receptor signal transduction . The cursh-fortilin) and random control sequence (lentivirussh-control) were purchased from Sigma . The lentivirussh-fortilin was experimentally shown to silence rat fortilin . Lentiviral particles that contained (a) the murine p53 promoter sequence fused to the GLuc cDNA, (b) the SEAP gene under the constitutional SV40 promoter, and (c) the puromycin resistance gene were obtained from GeneCopoeia  . The actual promoter sequence is as follows: GGATCTGTGGCTAGCTGGGGTTGGTCATCACCACCGCATGGCGGAGGCACCGGTTCAAAGTCTGTATTTTTCTCCGCTGGGGAACCTTGGGGTACCGGAGCTGGGGCCAGGTCAGGAGGGAGGCTATCCGGAGCTAAGAGTCGCTCCTCCGACGTCTTCATTCTGTAGAGTAAGCCCCCGGAAGGCAGAGGTCGGGCAAGTCTCGCTGAGCCGGCTACCAGCTGCCGAGGCTAGAGTGCATTACCGTTCCCAGGGATGCTCAGAGACCGGAGTCCGCTTTCCTCTTCCGGAAAATGTAAGCCGAACCTAAAGCAATCACCAGGGAACGAGTGTCCAAAGCCAAGCGCCTAGGGTCGCTAGGCGCCGCCAGGGCTTCTTGCTCTCGCGGGAGTCGGGCCACCTTCCGATAGGCTCTCCGCATCCTCCTCCGATTCCGAGCGGGAAGGCGGGAAGGAACGACTTTGCCTACACCTCAAGCGCTGGAGAATTCCTAGAGGTTTCTGGGAGTTGTAGTCTGAACTCTGGGCCTTGGCGAAAACTACACGAGCGCCCCCTACCGTCCCCTGGGGGTAATTCTTAAAGCGCCTATCCTCCCTGGCCTGCAGAGGGCGCATAATTTCTACAGTTTTTGCCCCTCTTGACTATCTTGTTTTGAATCCCKinase-Inhibiting RNase Attenuator 6 (KIRA6) , an inhiGTAACCTCAGGTTTCCTTTCTCCCCATCTCTCCCCCCTTCTTGTTCCTCTCTTTCCCTTTCTCCCCCGCCCTCCCTTCATTCATTCGACATTTATTATCAAGTTCTTACTGCCTAACCCAGGACTATACAAGGCATTGGGAAAAAAATAGCAATGTTTTCTAGTTCTTAATCTCCATAAAGTTTTCGTTGCTGTGCAATTAAAGGCTGTGAAAACAGTCTTTACAGAGAGTGATAAGGACTGTACAGGAAATTAAACACGGTGGTGCGATACCAAGTATCTCGGAGAACACGTTAGATTGAGATACTATGAAAAGCCTTTCTAAAGTGACATTTTAGCTAATGAGGGGAAAAAGAACTTAGGGGCCCGTGTTGGTTCATCCCTGTACTTGGAAGGCCTAAAGCAGGAAGACGGCCGCGAATTCCAGGCCAGCCTTGGCTACAAAGACTCTGTCTTAAAAATCCAAAAAGATGGCTATGACTATCTAGCTGGATAGGAAAGAGCACAGAGCTCAGAACAGTGGCGGTCCACTTACGATAAAAACTTAATTCTTTCCACTCTTTATACTTGACACAGAGGCAGGAGTCCTCCGAATCGGTTTCCACCCATTTTGCCCTCACAGCTCTATATCTTAGACGACTTTTCACAAAGCGTTCCTGCTGAGGGCAACATCTCAGGGAGAATCCTGACTCTGCAAGTCCCCGCCTCCATTTCTTGCCCTCAACCCACGGAAGGACTTGCCCTTACTTGTTATGGCGACTATCCAGCTTTGTGCCAGGAGTCTCGCGGGGGTTGCTGGGATTGGGACTTTCCCCTCCCACGTGCTCACCCTGGCTAAAGTTCTGTAGCTTCAGTTCATTGGGACCATCCTGGCTGTAGGTAGCGACTACAGTTAGGGGGCAC.® CRL-1446™) cell line (H9C2 hereafter) was directly obtained from American Type Culture Collection . Cells were maintained in Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum (FBS)  at 37 °C in an atmosphere containing 5% CO2. H9C2 cells stably harboring the lentiviral construct that contained both the p53 promoter sequence driving the expression of GLuc and the SEAP gene under the control of the constitutional SV40 promoter were generated by transducing the cells with Lentivirusp53-promoter-GLuc/SEAP and selecting them with puromycin (H9C2p53-promoter-GLuc/SEAP). They were maintained in puromycin-containing (0.3 µg/mL) media.MycoFluor™  was used to detect mycoplasma contamination when appropriate. The H9C2(2-1) (ATCCp53-promoter-GLuc/SEAP cells were transiently transduced by either (lentivirussh-fortilin) or (lentivirussh-control) at the multiplicity of infection (MOI) of 1. The next day, cells were irradiated using the RS-2000 X-ray irradiator  at 8 Gy. Seventy-two hours after the irradiation, conditioned media were collected and subjected to both SEAP and luciferase assays using the Secrete-Pair Dual Luminescence Assay Kit according to the manufacturer’s instruction (GeneCopoeia). The degree of activation of the p53 promoter was assessed by dividing the luciferase activity units by the SEAP activity units and expressing it in arbitrary units (A.U.).On the first day, H9C2Mice were given standard mouse chow  and water ad libitum, maintained on a 12-h light–dark cycle and seen by a scientist daily to observe their behavior and health. Any animal that displayed substantive signs of distress anytime during the protocol, including but not limited to poor grooming, hunched back posture, or a loss in weight exceeding 10% of the weights at the initiation of the experiments, were identified and treated accordingly, including removal from the experiment and euthanasia. For mouse experiments where grouping was based on pharmacological treatments, mice of the same genotype were randomly assigned to treatment and control groups.flox/flox mice, in which the fortilin gene was flanked by the LoxP sequence to allow tissue-specific deletion, were generated using the standard homologous recombination technique described previously: we micro-injected a mutant C57BL6 embryonic stem cell (ESC) line that contained two LoxP sequences flanking all six fortilin exons into C57BL6 blastocysts [flox/flox mice were fully in the C57BL/6J genetic background from the beginning. Fortilinflox/flox mice were then crossed with a transgenic flipase strain to remove the neomycin resistance gene cassette. To generate fortilinKO-heart mice, fortilinfloxflox mice were crossed with C57BL/6J mice overexpressing the Cre transgene under the control of cardiac-specific α-myosin heavy chain (Myh6) promoter [+/+, Jackson Laboratory, Ann Arbor, ME, Stock # 011038, already backcrossed to C57BL/6J for eight generations by the donating laboratory). Genotyping of fortilinKO-heart and fortilinWT-heart mice was performed on tail-derived genomic DNA using standard PCR-based methods. The presence of the αMHC-Cre transgene was detected using the following primer sets: 5′-ATGACAGAC AGATCCCTCCTATCTCC-3′ (M1) and 5′-CTCATCACTCGTTGCATCATCGAC-3′ (M2). αMHC-Cre+/− mice yielded a 300 bp amplified fragment and αMHC-Cre−/− mice yielded no amplicons. To determine the hemi- (αMHC-Cre+/−) or homo- (αMHC-Cre+/+) zygosity of the MHC-Cre transgene, RT-qPCR was performed using the primers and probe sets described previously [–ΔΔCT method [Forward primer (oIMR1084): 5’-GCGGTCTGGCAGTAAAAACTATC-3’Reverse primer (oIMR1085): 5’-GTGAAACAGCATTGCTGTCACTT-3’Probe (13593): 5’-FAM-AAACATGCTTCATCGTCGGTCCGG-IBFQ-3’FAM, 6-carboyfluorescein; IBFQ, Iowa Black™ double-quenched probe (Integrated Data Technologies (IDT), Coralville, IA)αMHC-Cre-transgene:Forward primer (oIMR1544): 5’-CACGTGGGCTCCAGCATT-3’Reverse primer (oIMR3580): 5’-TCACCAGTCATTTCTGCCTTTG-3’Probe (TmoIMR0105): 5’-JOE-CCAATGGTCGGGCACTGCTCAA-IBFQ-3’Apob (apolipoprotein B) [The target gene is otein B) .Internal control to calculate ΔCT for the αMHC-Cre-transgene:Fortilinstocysts . The respromoter  (αMHC-Creviously  (see belT method  to deterflox/flox mice yielded a 405 bp fragment and wild-type fortilin mice (fortilinWT/WT mice) yielded a 280 bp fragment.The presence of the LoxP-fortilin-LoxP knock-in construct was verified using the following primer sets: 5′-TGGACCCTGACTTTCATCACCTC-3′ (F1) and 5′- GTCATCTAACCTTACCCCAGTAAGC-3′ (F2): fortilinKO-heart mice were crossed with p53flox/flox mice available from the Jackson Laboratory . The status of the αMHC-Cre-transgene was evaluated by both PCR and RT-qPCR as described above, and the status of fortilin and p53 loci was determined using the following PCR-based genotyping strategies:Forward primer: 5’-TGGACCCTGACTTTCATCACCTC-3’Reverse primer: 5’-GTCATCTAACCTTACCCCAGTAAGC-3’Expected size of the mutant (floxed fortilin gene) allele: 405 bpExpected size of the wildp-type allele: 280 bpFortilin locus:Forward primer: 5’-GGTTAAACCCAGCTTGACCA-3’Reverse primer: 5’-GGAGGCAGAGACAGTTGGAG-3’Expected size of the mutant (floxed fortilin gene) allele: 390 bpExpected size of the wild-type allele: 270 bpP53 locus:To generate a strain of C57BL/6J mice that lack both fortilin and p53 in the heart, fortilinKOp53KO mice were daily and intraperitoneally injected with either 5 mg/kg of KIRA6 in solution  or the same solution without KIRA6 as vehicle (a) for the subsequent 10 weeks for the sacrifice group (N = 5 each) or (b) until death for the survival analysis group (N = 5 each). Mice were examined daily for their behavior and health. Mice in the sacrifice group were weighed and underwent transthoracic echocardiography before they were sacrificed, and their hearts were harvested and processed as described elsewhere in “Methods” section.For the KIRA6 rescue experiment, 8-week-old male fortilin2 and then by 0.5–3.0% isoflurane and placed on a heated platform, as described previously [KO-heart mice, the baseline echo was obtained when they were 7 weeks of age.Mouse transthoracic echocardiography was performed using a Vevo 2100 High-Resolution Imaging System  on mice anesthetized initially by 4% isoflurane in 1 L/min of Oeviously . We imagAfter being weighed, mice were sacrificed by isoflurane inhalation until effective, followed by exsanguination. Both the heart and lungs were routinely harvested. The liver, kidney, and spleen were harvested, when appropriate, for protein and RNA analyses. The harvested hearts and lungs were rinsed with phosphate-buffered saline (PBS), drained on absorbent paper, and weighed. The ratio of heart weight to body weight (HW/BW ratio) as well as that of lung weight to body weight (LW/BW ratio) was then calculated. For the sagittal cross-sectional analysis of the heart, the entire heart was fixed in 4% paraformaldehyde and subjected to paraffin embedding, sectioning, and hematoxylin and eosin staining according to the standard protocol. The right ventricle and atria were removed from the left ventricle (LV). The LV was then divided into three sections of equal size . The mid-ventricular part of the LV was fixed in 4% paraformaldehyde before being embedded in paraffin for immunohistochemistry. The apical portion of the LV was flash-frozen for subsequent protein extraction, and the basal portion of the LV was placed directly into Tri-Reagent  and frozen for subsequent RNA extraction.–ΔΔCT method [® RT-PCR kit  in the ABI Step One Plus Real-Time PCR system and the following primer and probe sets (Integrated DNA Technologies):Fortilin:Forward primer: 5′-TCCGACATCTACAAGATCCGG-3′,Reverse primer: 5′-ATCTTGCCCTCCACCTCCA-3′,Probe: 5′-FAM-AGATCGCGG/ZEN/ACGGGCTGTGC-IBFQ-3′ZEN™ = an internal quencher to enhance the quenching activity of the 3’ quencher IBFQ (IDT)Mouse Col1:Forward primer: 5′-GAAACCCGAGGTATGCTTGA-3′,Reverse primer: 5′-GTTGGGACAGTCCAGTTCTT-3′,Probe: 5′-FAM-TGTGCGATGACGTGCAATGCAATG-IBFQ-3′Mouse Myh7Forward primer: 5′-CCATCTCTGACAACGCCTATC-3′,Reverse primer: 5′-GGATGACCCTCTTAGTGTTGAC-3′,Probe: 5′-FAM-TCAGTCCATCCTCATCACCGGAGA-IBFQ-3′Mouse ANFForward primer: 5′-TCCGATAGATCTGCCCTCTT-3′,Reverse primer: 5′-CTCCAATCCTGTCAATCCTACC-3′,Probe: 5′-FAM-AAAGCAAAC/ZEN/TGAGGGCTCTGCTCG-IBFQ-3′Mouse Acta1Forward primer: 5′-CTCCCTGGAGAAGAGCTATGA-3′,Reverse primer: 5′-CGATAAAGGAAGGCTGGAAGAG-3′,Probe: 5′-FAM-CATCGGCAATGAGCGTTTCCGTTG-IBFQ-3′Mouse Serca2Forward primer: 5′-CATCAGTATGACGGGCTTGTAG-3′,Reverse primer: 5′-CTCGGTAGCTTCTCCAACTTTC-3′,Probe: 5′-FAM-AGCCACCATCTGTGCTCTGTGTAA-IBFQ-3′Mouse p53Forward primer: 5′-CAGCTTTGAGGTTCGTGTTTG-3′,Reverse primer: 5′-AGTTCAGGGCAAAGGACTTC-3′,Probe: 5′-FAM-TCTTCTTCT/ZEN/GTACGGCGGTCTCTCC-IBFQ-3′Mouse BaxForward primer: 5′-TTGCTGATGGCAACTTCAACTGGG-3′,Reverse primer: 5′-TGTCCAGCCCATGATGGTTCTGAT-3′,Probe: 5′- FAM-TTTGCTAGC/ZEN/AAACTGGTGCTCAAGGC-IBFQ-3′Mouse PumaForward primer: 5′-ATGGCGGACGACCTCAAC-3′,Reverse primer: 5′-AGTCCCATGAAGAGATTGTACATGAC-3′,Probe: 5′-FAM-AGCAGCATC/ZEN/GACACCGACCCTCAC-IBFQ-3′Mouse NoxaForward primer: 5′-TGCACCGGACATAACTGTGGTTCT-3′,Reverse primer: 5′-TGAGCACACTCGTCCTTCAAGTCT-3′,Probe: 5′-FAM-AAAGAGCAG/ZEN/GATGAGGAGCCCAAGC-IBFQ-3′Mouse 18S rRNAForward primer: 5′- GCCGCTAGAGGTGAAATTCT-3′,Reverse primer: 5′-TCGGAACTACGACGGTATCT-3′,Probe: 5′-JOEN-ACCAGAGCG/ZEN/AAA GCATTTGCCAAG-IBFQ-3′JOEN = 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluoresceinMouse RT-qPCR was performed as described previously . We usedT method  to calcuAnti-fortilin Anti-p53 Anti-GAPDH Anti-Bax Anti-PUMA Anti-NOXA Anti-Tropomyosin SDS–PAGE and Western blot analyses were performed as described previously , 37–40 oAll antibodies were used with appropriate IRDye680LT- or IRDye800CW-conjugated secondary antibodies . The signal intensities of protein bands were quantified using the Odyssey Infrared Imaging System (LI-COR) and normalized to the signal intensity of the loading control protein (GAPDH or tropomyosin) and expressed in A.U. To quantify p53 expression in response to X-ray irradiation, 0.5% (v/v) TCE was added to a polyacrylamide gel before polymerization. After standard SDS–PAGE, the gel was UV-irradiated on the Bio-Rad ChemiDoc MP Imaging System  for 2 min. The image was electronically captured, and the cumulative band densities were calculated to assess loading conditions as previously described . The sigTo evaluate the expression levels of fortilin in human hearts, we obtained de-identified tissue lysates of human hearts from patients with NFHs, non-ischemic cardiomyopathy (NICM), and ischemic cardiomyopathy (ICM) from the Duke Human Heart Repository . An automated capillary-based quantitative Western blot system called JESS™  was used to (a) detect fortilin  and (b) visualize total proteins loaded as described previously , 20. ComKOp53KO mice that were treated with either vehicle or KIRA6 were subjected to WES™ (Protein Simple), an automated capillary-based quantitative Western blot system, to detect p-IRE1α  and glyceraldehyde 3-phosphate dehydrogenase ; the latter served as the loading control. JESS™, but not WES™, can visualize the total proteins loaded for normalization. Compass Software (v3.1) was used to calculate a fortilin expression index by dividing the area under the curve of a fortilin peak by that of a GAPDH peak loaded in the same capillary . The fortilin expression index was expressed in A.U.To quantify phosphorylated IRE1α, the mouse heart lysates from fortilinCleaved lamin A . The cleavage of lamin is a well-characterized event in apoptosis .BAX p-IRE1α p53 phosphorylated p53 Mouse hearts were fixed in 4% paraformaldehyde and embedded in paraffin before they were sectioned at 5 µm thickness. Immunohistochemistry of mouse hearts was performed as described previously  using thTM DNA Fragmentation Detection Kit  following the manufacturer’s instructions. TUNEL-positive cells within about 0.145 mm2 of the ROI were counted, and TUNEL indices were calculated as the number of TUNEL-positive cells per unit area (in mm2). Results are expressed in A.U.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of heart tissue was performed on formalin-fixed, paraffin-embedded samples as previously described , 46 usinTo assess the presence of cardiomyocyte hypertrophy, the heart tissue that had been formalin-fixed and paraffin-embedded was sectioned at 5 µm thickness and stained with laminin . After stained sections were digitally captured using the EasyScan Digital Slide Scanner, cardiomyocyte areas were measured using ImageJ and expressed in A.U. as described previously . At leasThis study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments involving animals were approved by the Institutional Animal Care and Use Committees of our institution. Human tissue samples used for this study were procured from the Duke Human Heart Repository (DHHR), which is a Duke University Health System Institutional Review Board (DUHS IRB) approved tissue repository. Samples were procured by the DHHR using written informed consent or a waiver of consent for discarded tissues. No HIPAA information was provided with any of the samples used in this study. Human myocardium was acquired from the left ventricular free wall of explanted hearts following cardiac transplantation. Non-failing (NF) left ventricular tissue was acquired from donors whose hearts were not utilized for transplant, thus becoming available for research.N) is indicated in either figures or the main text. The degree of the spread of data was expressed by the standard deviation (±SD). The difference between the control and study groups was analyzed using unpaired two-tailed Student’s t-test for two groups or one-way analysis of variance (ANOVA) followed by Fisher’s pairwise multiple comparisons for multiple groups. P < 0.05 was considered to be statistically significant. For survival analyses, Kaplan–Meier survival curves were generated, and the Log-Rank (Mantel–Cox) test was used to compare the curves. The numbers of mice used in in vivo experiments were determined by (i) power analysis, assuming an α error rate of 0.05, a β error rate of 0.20, and an expected difference of 25%, in Minitab 17  or (ii) our previous dataset and experience from similar experiments performed as part of past research. A similar variance was observed between the groups that were statistically compared. No data were excluded unless outliers were identified and verified by the outlier tests (Minitab 17). Although the scientists were not blinded to allocation during experiments and readouts evaluation, all readouts from the experiments were predetermined, highly objective, and obtained according to the validated protocols.All measurements were taken from distinct samples  and the size of biological samples (SUPPLEMENTAL MATERIAL


Surveying transcriptome-wide Igf2bp2 client mRNAs in white adipocytes reveals selective binding to mRNAs encoding adipogenic regulators and energy expenditure effectors, including adiponectin. These same target proteins are downregulated when either Igf2bp2 or lncRAP2 is downregulated, hindering adipocyte lipolysis. Proteomics and ribosome profiling show this occurs predominantly through mRNA accumulation, as lncRAP2-Igf2bp2 complex binding does not impact translation efficiency. Phenome-wide association studies reveal specific associations of genetic variants within both lncRAP2 and Igf2bp2 with body mass and type 2 diabetes, and both lncRAP2 and Igf2bp2 are suppressed in adipose depots of obese and diabetic individuals. Thus, the lncRAP2-Igf2bp2 complex potentiates adipose development and energy expenditure and is associated with susceptibility to obesity-linked diabetes.lncRAP2 is a conserved cytoplasmic lncRNA enriched in adipose tissue and required for adipogenesis. Using purification and   •lncRAP2 is a cytosolic lncRNA conserved in mouse and human needed for adipogenesis•lncRAP2 complexes with mRNA stability and translation regulators, including Igf2bp2•lncRAP2-Igf2bp2 stabilizes lipid metabolism mRNAs to potentiate energy expenditure•lncRAP2-Igf2bp2 genetic and expression variation is linked to BMI, type 2 diabetes Biological sciences; Molecular physiology; Molecular biology; Omics Previous studies revealed that many lncRNAs are specifically enriched in white and/or brown adipocytes and play vital roles in adipocyte biology , includiin situ hybridization (smFISH) visually determines RNA abundance and location, revealing whether a lncRNA diffuses to trans sites beyond its chromosomal locus or remains tethered in cis in the nucleus (in vivo (rather than in solution after cell lysis)  . And fool lysis) .Here, we use these and other tools to interrogate the mode of action of lncRAP2, a white adipocyte-selective RNA that is essential for adipogenesis . We showin situ hybridization (smFISH) reveals that lncRAP2 diffuses from the nucleus, spreading throughout the cytoplasm at 14 ± 3 transcripts per white and 9 ± 3 transcripts per brown adipocyte   in 3T3-Lvia posttranscriptional control of metabolically important proteins, impacting sensitivity to diet-induced obesity and type 2 diabetes risk  (Igf2bp2 with those of depleting Pparγ or linc-ADAL (lincRNA for adipogenesis and lipogenesis) in mature white adipocytes  D and S3Fipocytes   , including 138 whose mRNAs were found to be direct Igf2bp2 targets by RIP-seq. For both direct Igf2bp2 targets and non-targets, the changes in protein levels occurring upon lncRAP2 depletion closely correspond to those occurring upon  t test) F. These depleted A, such adepleted G. By conIgf2bp2 depletion in mature adipocytes for 1,205 genes quantifiable by both RNA-seq and mass spectrometry. The correlation between the two types of responses was strong for direct Igf2bp2 targets with differentially regulated protein vs. non-targets, both after lncRAP2 depletion  and after Igf2bp2 depletion   I and S4B t test) . We founepletion J. In sum2 ≥ 0.8) with variants within lncRAP2, but not so with any other locus within 500kb, including the IDE gene  associated with increased body mass index, higher fasting insulin levels, and insulin resistance in women with polycystic ovary syndrome in a Chinese population  maps to IDE gene A and S5AIDE gene A, insulaIDE gene B.Figure −8). Additional genetic variants mapping to the lncRAP2 transcription start and end sites  and gene body (rs967878) also represent risk alleles for type 2 diabetes (p < 109 to 10−8), thus evidencing association of multiple lncRAP2 alleles with diabetes  (Igf2bp2 levels decrease as body mass index increases (Igf2bp2 trends lower in adipocytes of leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) obese mice  (The pathogenesis of type 2 diabetes can involve obesity-linked adipose dysfunction . To expl t test) E. Human ncreases D and S5Encreases F. We alsese mice F, which ese mice . We spec, ttest) G. Thus, Obesity has become pandemic , increasOur data show that multiple genetic variants associated with propensity to type 2 diabetes are confined to an lncRAP2 linkage disequilibrium and chromatin interaction domain. Analyzing hundreds of phenotypes interrogated by genome-wide association studies reveals that these variants, mapping to the lncRAP2 gene body and transcription start and end sites, are specifically associated with body fat mass, insulin secretion, and type 2 diabetes, implicating lncRAP2 alleles in the development of obesity and diabetes. Supporting this notion, lncRAP2 is suppressed in the white fat of mice and humans consuming a high-fat diet, and in humans lncRAP2 levels decrease progressively with increasing obesity, whereas lncRAP2 levels are restored upon weight loss.lncRAP2 is enriched in white relative to brown adipocytes and is critical for adipogenesis . AlthougIgf2bp2 levels in pancreatic islets .Further information and requests for resources and reagents should be directed to the lead contact, Marko Knoll (This study did not generate new unique reagents.C57BL/6J mice were bred in house or purchased from Jackson Laboratories (stock # 000664). All mice were housed under a 12 h light/dark cycle at constant temperature (20°C). All procedures were performed according to protocols approved by the Committee on Animal Care at the Massachusetts Institute of Technology.2 asphyxiation and interscapular brown adipose tissue (BAT) and subcutaneous  white adipose tissue (scWAT) was harvested into room temperature plain DMEM . The fat pads were transferred into a well of a 6 well plate and minced with scissors for 5 min. Minced tissues were then transferred into a 50 mL conical tube with 3 mL Hank's balanced salt solution  supplemented with 0.2% collagenase A  and 2% BSA  using a 1 mL pipet tip with the tip cut off to allow aspiration of larger pieces. The tissues were incubated agitating (350rpm) and repeated vortexing every 5 min for 10 s at 37°C for 30 min or 20 min for scWAT. Following collagenase digestion, 10 mL room temperature plain DMEM was added and cells were filtered through a 70 μm mesh filter . Mature adipocytes and the stromal vascular fraction (SVF) were separated by centrifugation at 700 g for 5 min. The supernatant was removed and the SVF resuspended in 10 mL room temperature plain DMEM followed by additional filtering through a 30 μm mesh filter (Miltenyi Biotec # 130-041-407) and subsequent centrifugation at 700 g for 5 min. The SVF from subcutaneous white fat pads (scWAT) of 8 mice were then resuspended in 10 mL DMEM supplemented with 10% heat inactivated new born calf serum  and plated on two 10 cm dishes . The SVF from interscapular brown fat pads of 6–8 mice were then resuspended in 6 mL DMEM supplemented with 10% HI NCBS and plated on 3 wells of a six well plate . After 4 and 24 h, the medium was replaced by fresh, pre-warmed DMEM/10% HI NBCS at 37°C and with 5% CO2. Cells were grown to confluence and then passaged no more than two times before seeding the pre-adipocytes for differentiation.6–8 male 2–4 week old mice were sacrificed by COPre-adipocytes derived from BAT were cultured to confluence and then subsequently overgrown for 4–6 additional days until growth arrested. The cells were then induced to differentiate by culturing them for two days in induction medium consisting of DMEM supplemented with 10% fetal bovine serum  and 850 nM insulin (Sigma-Aldrich #I1882), 0.5 μM dexamethasone (Sigma-Aldrich #D4902), 250 μM 3-isobutyl-1-methylxanthine , 1 μM rosiglitazone , 1 nM 3,3,5-triiodo-L-thyronine  and 125 nM indomethacin (Sigma-Aldrich #I7378). Subsequently, the induction medium was replaced with DMEM supplemented with 10% FBS and 160 nM insulin and 1 nM T3 for another two days. The cells were then cultured in DMEM 10% FBS and 1 nM T3 until day 8 of differentiation, and the medium was replaced every other day. Pre-adipocytes from scWAT were cultured similar but the induction medium and following medium did not contain T3.Mature adipocyte cell layers were washed twice in plain pre-warmed DMEM and stimulated with 1 μM isoproterenol (Sigma-Aldrich I6504). After 6 h of stimulation, the cells were washed once with cold PBS and RNA was harvested using TRizol or QIAzol lysis reagent as described below. For immunoblotting, the cultures were harvested after stimulation with isoproterenol after washing with cold PBS on ice and adding 40 μL RIPA buffer per well of a 6 well plate or 100 μL RIPA buffer to a 10 cm dish.Was performed as described . BrieflyFollowing addition of fresh medium, cells were stimulated with isoproterenol. Cell culture medium was collected after 24h of stimulation and stored at−20°C. Glycerol release was measured using the Adipolysis Assay Kit  following the instructions of the manufacturer.Total RNA was isolated from tissues or cells using TRizol or QIazol reagent (LifeTechnologies/Ambion) and a miRNAeasy kit (Qiagen). 300 ng were reverse transcribed using Superscript II reverse transcriptase (LifeTechnologies/Invitrogen) using random hexamers (LifeTechnologies/Invitrogen). The cDNA was diluted 1:10 and 2.5 μL for a 96 well plate or 1 μL for a 384 well plate were used for quantitative Real-time PCR. qPCR was carried out on an ABI7900HT Fast real-time PCR system (Applied Biosystems) and analyzed using the delta delta Ct method normalized to 18S if not stated otherwise. Results are shown as pooled data from 3–4 independent experiments displaying the average and SEM.Poly A+ RNA sequencing (TrueSeqStrandedPolyA) was performed on RNA samples using a Nextseq genome sequencer (Illumina). RNA-seq paired-end reads were aligned to the mouse genome  using STAR v2.6.1  providedLysates were centrifuged for 10 min at 13,000 g to remove debris, and NuPAGE sample buffer and reducing buffer (LifeTechnologies) were added after measuring and adjusting the samples for protein concentration . 2–20 μg protein per sample were separated for 2–4 h at 60–100 V using 8% 26 well NuPAGE Bis-Tris Midi gels in MOPS or MES buffer (LifeTechnologies) and in Criterion cells (Bio-Rad) using respective adapters (LifeTechnologies). Protein was wet transferred to polyvinylidene fluoride (PVDF) membranes  in Criterion blotter cells (Bio-Rad) using 2 x NuPAGE Transfer buffer (LifeTechnologies) with 10% methanol for 25 min at 1 A. After blocking the membrane in filtered  TBS-T  with 3% BSA  for 1 h, blots were incubated in primary antibody diluted in TBS-T 3% BSA sealed in hybridization bags and gently shaking at 4°C overnight, then washed three times for 10 min in TBS-T, incubated in secondary antibody  diluted in TBS-T 3% BSA gently shaking for one hour at room temperature, and then washed again three times for 10 min in TBS-T. Antibody binding was visualized using ECL Plus Western Lightning reagent (PerkinElmer NEL 102) and blots were exposed to film . Films were scanned without adjustments using an Epson scanner. All immunoblotting data shown were reproduced with almost identical results in at least one and typically two to three additional independent experiments.Cells were washed in PBS and fixed in 3.7% formaldehyde solution for 1 h, followed by staining with Oil Red O for 1 h. Oil Red O was prepared by diluting a stock solution (0.5 g of Oil Red O (Sigma) in 100 mL of isopropanol) with water (6:4) followed by filtration. After staining, plates were washed twice in water and photographed.The 5′ and-3′ ends of lncRAP2 was determined using the FirstChoice RLM-Race Kit from Ambion following the manufacturers instructions. Primers were designed accordingly and can be found in To separate the nuclear from the cytoplasmic fraction differentiated 3T3-L1 adipocytes were harvested and 1 million cells were used to isolated the fractions using the PARIS kit (Life technologies) according to the manufacturers instructions. Separated fractions were then analyzed using Real-time PCR and gene specific primers.Single molecule RNA FISH and fluorescence microscopy were described previously . Brieflyhttp://hannonlab.cshl.edu/fastx_toolkit/index.html), discarding non-clipped reads or reads <25nt after linker clipping (“-l 25 -c” parameters), andRibosome profiling sequencing reads were clipped to remove 3′ linkers using fastx_clipper from the FASTX-Toolkit  using STAR v2.6.1 with default parameters and “--seedSearchStartLmax 20 --sjdbOverhang 39 --outSJfilterOverhangMin 30 8 8 8 --sjdbScore 2”. To measure gene-level and region-level  expression from uniquely-aligned reads, we quantified read counts using HTseq-count. Only genes for which a read count could be obtained in each sample and replicate were retained, and counts were then normalized as counts per million mapped reads (cpm) using only reads from these genes.Gene-level translation efficiency was calculated as the ratio of normalized Ribo-seq read density (cpm) to the normalized RNA-seq read density (cpm).We computed the ribosome release score using the RRS program  default. For mRN7 re-suspended in 2mL 1X PBS, then lysed in nuclear isolation buffer  for 20 min. Nuclei were pelleted by centrifugation at 2,500 g for 15 min. Supernatant was discarded and nuclei were re-supended in 1mL RIP buffer containing the HALT protease and phosphatase inhibitor (Thermo scientific) and split into two fractions and mechanically sheared using a Dounce homogenizer with 20 strokes. Nuclear membrane and debris were pelleted by centrifugation at 13,000 rpm for 10 min at 4°C. The supernatant was pre-cleared by adding 30μL slurry of protein A/G beads  and incubation for 2 h at 4°C on a rotator. Beads were removed by centrifugation at 2500 g for 1 min and 10% of the supernatant was removed to a new tube (10% input) and the rest was incubated with antibodies to Suz12 , IgG , Ezh2 , hnRNPU , CoREST  or no antibody for 3h at 4°C on a rotator. Then 60μL slurry of protein A/G beads were added for 2h at 4°C on a rotator. Beads were pelleted by centrifugation at 2500 rpm for 30s and washed 3 times in 500μL RIP for 10 min each followed by one wash with 1X PBS. For the isolation of RNA, the beads were re-suspended in TRIzol after the last wash step and isolated according to the manufacturers instructions. The RNA pellet was re-suspended in 10μL dH2O and was directly used for reverse transcription using random hexamers and SuperScript II (Invitrogen). Analysis was done by real-time PCR and sequencing.RNA coimmunprecipitation was done as described . Mature RNA FISH probes were biotinylated using Biotin-XX, SSE (Thermo Fisher). As controls, we also used probes against H19  and BlooRaw reads (40bp unpaired and non-strand-specific) were mapped to mm9 using bowtie2 . Peak caChIRP-MS was performed as described  with theMass spectrometry fragmentation spectra were correlated against a custom peptide database, formed by downloading all RefSeq species-specific (mm9) entries, and against a database of common contaminants  using Mascot (Matrix Science) 2.5.1 and the Sequest algorithm (Thermo). The resulting Mascot search results were uploaded into Scaffold (Proteome Software), and a minimum of two peptides and peptide threshold of 95% and protein threshold of 99% were used for identification of peptides and positive protein identifications. Proteins enriched in ChIRP-MS experiments were identified based on several criteria. First, only proteins identified by 2 or more unique peptides in the differentiated sample were considered. Then proteins whose total peptide count was at least 10-fold greater in the exon probes versus intron probes were considered enriched. Finally, proteins introduced during sample preparation and purification  were excluded from further analysis. To distinguish lncRAP2-specific interactors from proteins that generally bind RNA during its processing, we compared the proteins enriched by lncRAP2 with those enriched by Dleu2 , Neat1 aAfter proteins were separated on an Acryl-amid gel (see Western blot), the gel was fixed in 40% EtOH with 10% HAc for 1 h then washed 2 × 20 min in 30% EtOH and once for 20 min in water. The gel was sensitized for 1 min in 0.02% Na2S2O3 following 3 × 20 s wash in water and 20 min incubation in cold (4°C) 0.1% AgNO3 solution. The gel was washed 3 × 20 s in water and developed in a new chamber in 3% Na2CO3 with 0.05% formaldehyde. As soon as bands became visible the reaction was stopped by a short wash in water and then incubation in 5% HAc. All lanes were cut out and processed .Gene lists were analyzed for enrichment of genes grouped by biological process ontology or by curated annotations from the Molecular signatures database with GSEA  using deGenomic sequences from regions of interest (e.g. UTRs) were searched for matches to a database of TF recognition sites  for TFs Processed ChIA-PET, Hi-C, and capture Hi-C datasets were downloaded and visualized in triangle heatmap mode using the UCSC genome browser or in arc mode using the Washington University Epigenome browser, with default normalization and resolution settings and fixed display values.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Genome-wide read density maps were generated by MACS2 using the “--bdg" option, normalized by RSeQC (http://CRAN.R-project.org/package=gplots).All sequencing reads were quality-checked with FastQC  was assessed by a Fisher's exact test using the fisher.test R implementation with default parameters. For unpaired data, a Shapiro-Wilk normality test was first performed using the shapiro.test R implementation with default parameters; for normally distributed data we then used a two-sided ttest (t.test R implementation with default parameters) to assess confidence on the measured difference of their mean values. For unpaired data that don't follow a normal distribution, we used a non-parametric Wilcoxon rank-sum test to determine if they belong to the same distribution. Variance was represented as mean ± SEM of n = 3 replicates unless otherwise specified.>hg19_chr10: 94,176,182-94,180,397ATCCGTTACCCAACGGCTGTCGAAAGAGGAGCACGACGTGAACTTCCACCGACAATCGTTTAAATACTGCAGGCGAAGGACGGGTTCTTATTGTTCCTTCAGGCAAACCGGCAGCTGAGTCCAGCTTGTGAACTTGAACTTGAACTTGCTGAAGAAGCTCCCGGCGGCCCTCTGCTGGCCGCCGCCTTTGCCAGAGGGAGAGGCTGGAGTCACCCTGTTGGAACCCATTTCTGGGGCTGGCACCTGTTGGGCTGCCCGGCCGCGCGTACCTGGTCCCATCGGGGGCTCTGCCCACTCCGCTGATGACGCGGGTAGAAGGGAGGCCGCAGGGACACTCTGGGGGGACTGTGCCGGGCGGGCACCCCCCAGCTGCTCACTGTGGGGTGCGGCACCGAGGCCTGGTTGGGCTGCAAGGAGACCGACTGGGATTCCCGGGCTGGTGGCCGGGGAGACGGGGTAGAGGTGAGAAGCAAGAGCTCAGGAGGCCTCAGGCCCCAGCGCTGTGGGGCTGCCGTTGTCGTTCTGGGTGGAGGTCTGGCCAAACCGGCTTTTGCCCCGAGTGAGGAATTCCTGCTCATTTTGGTGTTAGTGGAGAGGTCGCTGTTCACAGCGGGGGTGGGGTTCGTCCCCTCCAGGCTAGTAGGGAGCTGGCTGGTGCATTGCTGTGTTGCTGCCCTTCTGCCCTGTCTCCTGATCCTGTTCAGACTCTGGGTGGCTCCTTTGAGTCTTCTCAGGCCGTGAAAGATGGGAAAGGCAGGTTGTTGAAACCACTGCTGCTCCGGGCTGGCAGAGACCCTGAGCTGTACCTCCTGGGATGAAAGGAGAACCCAGGAGCAGGCAGGTGACCACTGCTTCCTCATACTCCTCAGCAAGTCCTGGATCTCTTCCTGTCTGTAACCCACGGATACCATGAACTTAGTTTGCAGGGGGTCCTCCCAGTGGGAGAGTGGCTGCACACAGCGCTTAAGTTCCTCACCTTCATGACTCGCATTCATCCATGGACCCCTCACAGTGTGCTCTGAAGGGCCTCTCTTCCTGCAATTGACAATGAGAAATTTCTTCAGCAAGTTTTCACTCTCCGGGGACATGGGGGAGGGAATGGAATACATCCTGCTCAGTACCTGCTCCTATAGCTCCTTGAAGTTCTGTCCATCGAAAAATAAGGATCCATTTACCAGTATATAGAGGATAACTCGCAGACGCCATACATCCACCAGCTCATATTTGTGGCCCTAGAAGAATTCCAGGGCAGCCTAAGGGGACTGCCACAAAAGGTATCCAGCTTGTTGCGAAAGGCGAATTTATTCCTGAACCCAATCTGTGATGTTCATGTTAACATGGACAATACATTTCTGGTGACAATACACAATATCTGTGGACAATGCACTTCTGGTAACAGCTTGCATAGCAGACACTTTTGGTGGAATTTGCTTCAGGCCTCTTTCCTGCTGCTGTGAGCAGGTATTCACAAACTTCTCGCTAGCGTGCTTTGTGATGAGGCAGAGATTCCTCACTATCGATCACAGCAAATACTTCACTATGTTGGGGTGATCTGAGCCTTCATGATTCCTACTTTGTGGATGGTGTCTGGAGGCTGGAGGAGATCTGCTGAGTCTCATCAATGAACTTCACGGCGGCTGCTTTCCCAGTCAGGATGTCCCAGGCCAACCCCACCTTAGCCAGGTTGTCCTTGACGATGGTCTCAAGGATCCTCGCTGCCAATAAGGATCCCTCCTCAGCAGAGATGGCTGAGAGGCCCTGCGGCGTGTGGGACTTACTACTGGGCTTGGAGTCAAGGTGTCCCAGGTCGGCTTAATTTTGGGAAAAGCTGGTGAGAAGCTTGGTCCAAATCACCTGAAAAGAAAATCACTTGCATAGTTTACCTGAAGAACGGATTTCTATAATGGAATCAAAACACAATACTGGACAAAAATCAAATAAGCAAAAGCAACAAACTGAAAAGGAAATGCTGAGTAAAAAAGAACTTAAAAATTAGAAAAATAAGAGAAAAAAGAACAAGAAGAACCAGTAACCTCAGCCAAGCCAGGGACTTACATACGTGCTTGGAATTCCAAAGCAGCAGTTCCTTATGAGGAAGAACTAAGCCTAGTAACAAGGCTGAGGATAATCTATGTGGCTTTCTCATGCTTTGGTCTCAAGAACTCTTTACTCTTAAAGAAAATATATTGAGGACCACAAAGAGGATTTTTTATTGATATGGGTTACAGTTATGAATATTTACCTTATTAGAAATTAAAACCTCTAGGATGCTTCAATGGCCTTTTCTAGTTTGAAAAGATAACAGGCTGGGTGTGGTGGCTCACGCCTGTAATTCCAGCACTTTGGGAGGCCGAGGTGGGCAAATCACCTGAGCTCGGGAGTTCGAGAAAAGGTATAAAAATGTTTGGCTTTTAAAGAGCCCACAATATCTACACTTAAAATATTTCATTTTTTTCTTTAAACTCTAAATGATTGGTTTCAAAATGATGCCACAACTTAGCTGGCATTATGATAGTGTATAAGTATGTTCTGTTGTGTACGACACAATGAGCTTCATTATTACCATCTGCGACACTGAGGGGAAGATAGCTTTCATCATATTTTCTCATTTAAAGTTTTGCCCAGTTTCATTTGCATAGATTCCCTTTTTCCATGAGCTGCTATGTCAGTCTCAGCATCTTTCAATGTAGAGTTTGCAGCTATGAGTTGAGAAAGCACATTTTCTACTCTTTTTAAGTGAATAATCCACTGTGCCTGGTGTACCTCTCCTTCAGCATAGGATAGGGACATCCAGGTACTGGACCCGTCACTGGCACCTCAGTGGGGAGAACCCAGATGCCCCTACATGATGTTTAAAGATGCTTTATATACATAAAAGTGCACAAATCATCAGCCCACAGCTTGGTGACTGTTCACATATTGAACTCATCTATTTATCTAGTATCCAGGTCAAGAAACAGCCATTACAGCCCCCCAAGATCCCACACCCCTTTTCCAGTCACTTTCTCTGCAGTGATAACCACTCTTCTGTATTTTGACAGCATAGATTCATTTTGCTTATTTTTGAACTTTACATACATGGATTCATACAGTATTGGATCCTTTGTGTCTGCTTCCTTTGCTTAATTTGTTTTTGTTTGTTTGTTTGTTTGTTTGTTTTTCTTGAGACAGAGTCTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCACGATCTCAGATCACTGCAACCTCCACCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTATAGGAGCTTGCCACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTTACCTCATGTTCCGCCTGCCTCAGCCTCCCAAAGTGCTGGAATTACAGGCATGAACAACCACGCCTGGTCCTTTGCTCAATATTTTTGTGAGATCCATCCATATTGTTTATTATTCTAAATGCTCATTGTGTGACTGTAACACAATTTGTTAATTTGTTTATTCATTTTACTGTTACTGGGCAGTTGAGTAGTTCTCAGTTTTCAGATGCTATAGTGCTGCCATAAACATTCTTGTTCAGGTTTTTGGGGGACATATATATGGCTTTCTGTTGGATATATATAAATATATTAAGGGTGTGGCTGAACAACCATTTGACAGTTTATGCTAACAAGGTGACTCGTGGTAGGCCCCTTAGGCCAGGTGATATCAGCCTGACCTCCAGAGAGTGGGGTGGGGGCTGGAGACTGAGTTCAACCACATGGACAATAAGTCTATCATGTAATGAAGCCCCAGTAAAAACTCTGGATGCTGAAGCTCAGGTGAGTGTCCCTGATTGGCAGTACTCTATATGTGTTGTCTCACACATCCAAATCAGCAGGGTAATGCATTCTGAGGACCCCAGAGGCTTCACATTTGGAACCCTCTCAGACTCTGCTCTATCAATCTCTTTCTTTGGCTAATTTTGATCTCTATCCTTTCCCTGAAATAAACTGTAACTGTGAGTATAACAG.


Messenger RNA (mRNA) comprises three major parts: a 5'-UTR (UnTranslated Region), a coding region, and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Some sequences in the 3'-UTR are involved in the localization of mRNAs in (e.g.) neurons, epithelial cells, oocytes and early embryos, but such localization has been most thoroughly studied in neurons. Neuronal polarity is maintained by the microtubules (MTs) found along both dendrites and axon and is partially influenced by sub-cellular mRNA localization. A widely studied mRNA is that for Tau protein, which is located in the axon hillock and growth cone; its localization depends on the well-characterized cis-acting signal (U-rich region) in the 3'-UTR.We compared the cis-acting signal of Tau with mRNAs in the axonal regions of neurons using the ClustalW program for alignment of sequences and the Mfold program for analysis of secondary structures.We found that at least 3 out of 12 mRNA analyzed  have a sequence similar to the cis-acting signal of Tau in the 3'-UTR. This could indicate that these messengers are localized specifically in the axon. The Mfold program showed that these mRNAs have a similar "bubble" structure in the putative sequence signal.Hence, we suggest that a U-rich sequence in the 3'-UTR region of the mRNA could act as a signal for its localization in the axon in neuronal cells. Sequences homologous to the DTE sequence of BC1 mRNA could direct the messenger to the dendrites. Messengers with homologues of both types of sequence, e.g. β-actin, might be located in both dendrites and axon. A messenger RNA (mRNA) comprises three major parts, a 5'-UTR (UnTranslated Region), a coding region and a 3'-UTR. The 3'-UTR contains signal sequences involved in polyadenylation, degradation and localization/stabilization processes. Many studies have shown that certain sequences in the 3'-UTR are involved in localizing the mRNAs in different cells such as neurons, epithelial cells, oocytes and early embryos ,2. Such The mRNA of tau has been studied in detail . Tau is Recently, many mRNAs have been shown to be located in neuronal axons: β-actin, tropomyosin 3 (Tpm3), cofilin, vimentin, immunoglobulin heavy chain biding protein (Bip), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose regulated protein (grp75) and synuclein . The objThe 3'-UTR of tau mRNA contains 3884 bases; the U-rich region (in bold) is responsible for the localization of this mRNA in the axon hillock and growth cone.UUUUUUUUUUUUUACUUUAGCGGUUGCCUCCUAGGCCUGACUCCUUCCCAUGUUGAACUGGAGGCAGCCAAGUUAGGUGUCAAUGUCCUGGCAUCAGUAUGAACAGUC AGUAGUCCCAGGGCAGGGCCACACUUCUCCCAUCUUCUGCUUCCACCCCAGCUUGUGAUUGCUAGCCUCCCAGAGCUCAGCCGCCAUUAAGUCCCCAUGCACGUAAUCAGCCCUUCCAUA CCCCAAUUUGGGGAACAUACCCCUUGAUUGAAAUGUUUUCCCUCCAGUCCUAUGGAAGCGGUGCUGCCUGCCUGCUGGAGCAGCCAGCCAUCUCAGAGACGCAGCCCUUUCUCUCCUGUC CGCACCCUGCUGCGCUGUAGUCGGAUUCGUCUGUUUGUCUGGGUUCACCAGAGUGACUAUGAUAGUGAAAAGAAAAAGAAAAAGAAAAAAGAAAAAAGAAAAAAAAAAAAGGACGCAUGU UAUCUUGAAAUAUUUGUCAAAAGGUUGUAGCCCACCGCAGGGAUUGGAGGGCCUGAUAUUCCUUGUCUUCUUCGUGACUUAGGUCCAGGCCGGUCGAGUGCUACCCUGCUGGACAUCCCA UGUUUUGAAGGGUUUCUUCUUCAUCUGGGACCCCUGCAGACACUGGAUUGUGACAUUGGAGGUCUAUACAUUGGCCAAGGCUGAAGCACAGGACCCGUUAGAGGCAGCAGGCUCCGACUG UCAGGGAGAGCUUGUGGCUGGCCUGUUUCUCUGAGUGAAGAUGGUCCUCUCUAAUCACAACUUCAAGUCCCACAGCAGCCCUGGCAGACAUCUAAGAACUCCUGCAUCACAAGAGAAAAG GACACUAGUACCAGCAGGGAGAGCUGUGGCCCUAGAAAUUCCAUGACUCUCCACUACUAUCCGUGGGUCCUUUCCAAGCCUUGCCUCGUCACCAAGGGCUUGGGAUGGACUGCCCCACUG AUGAAAGGGACAUCUUUGGAGACCCCCUUGGUUUCCAAGGCGUCAGCCCCCUGACCUUGCAUGACCUCCUACAGCUGAAGGAUGAGGCCUUUAAAGAUUAGGAACCUCAGGCCCAGGUCG GCCACUUUGGGCUUGGGUACAGUUAGGGACGAUGCGGUAGAAGGAGGUGGCCAACCUUUCCAUAUAAGAGUUCUGUGUGCCCAGAGCUACCCUAUUGUGAGCUCCCCACUGCUGAUGGAC UUUAGCUGUCCUUAGAAGUGAAGAGUCCAACGGAGGAAAAGGAAGUGUGGUUUGAUGGUCUGUGGUCCCUUCAUCAUGGUUACCUGUUGUGGUUUUCUCUGUAUACCCCCAUUUACCCAU CCUGCAGUUCCUGUCCUUGAAUAGGGGUGGGGGUACUCUGCCAUAUCUCUUGUAGGCAGUCAGCCCCCAAGUCAUAGUUUGGAGUGAUCUGGUCAGUGCUAAUAGGCAGUUUACAAGGAA UUCUGGCUUGUUACUUCAGUGAGGACAAUCCCCCAAGGCCCUGGCACCUGUCCUGUCUUUCCAUGGCUCUCCACUGCAGAGCCAAUGUCUUUGGGUGGGCUAGAUAGGGUGUACAAUUUG CCUGGAACCUCCAAGCUCUUAAUCCACUUUAUCAAUAGUUCCAUUUAAAUUGACUUCAAUAUAAGAGUGUAUCCAUUUGAGAUUGCUUGUGUUGUGGGGUAAAGGGGGGAGGAGGAACAU GUUAAGAUAAUUGACAUGGGCAAGGGGAAGUCUUGAAGUGUAGCAGUUAAACCAUCUUGUAGCCCCAUUCAUGAUGUUGACCACUUGCUAGAGAGAAGAGGUGCCAUAAGGCUAGAACCU AGAGGCUUGGCUGUCCACCAACAGGCAGGCUUUUGCAAGGCAGAGGCAGCCAGCUAGGUCCCUGACUUCCCAGCCAGGUGCAGCUCUAAGAACUGCUCUUGCCUGCUGCCUUCUUGUGGU GUCCAGAGCCCACAGCCAAUGCCUCCUCAAAACCCUGGCUUCCUUCCUUCUAAUCCACUGGCACAUCAGCAUCACCUCCGGAUUGACUUCAGAUCCACAGCCUACACUACUAGCAGUGGG UAAGACCACUUCCUUUGUCCUUGUCUGUUCUCCAGAAAAGUGGGCAUGGAGGCGGUGUUAAUAACUAUAGGUCUGUGGCUUUAUGAGCCUUCAAACUUCUCUCUAGCUUCUGAAAGGGUU ACUUUUGGGCAGUAUUGCAGUCUCACCCUCCGAUGGCUGUAGCCUGUGCAGUUGCUGUACUGGGCAUGAUCUCCAGUGCUUGCAAGUCCCAUGAUUUCUUUGGUGUUUUGAGGGUGGGGG GAGGGACAUGAAUCAUCUUAGCUUAGCUUCCUGUCUGUGAAUGUCCAUAUAGUGUACUGUGUUUUAACAAACGAUUUACACUGACUGUUGCUGUACAAGUGAAUUUGGAAAUAAAGUUAU UACUCUGAUUAAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCAGGCCCCUGGGGCCGUCACUGAUCAUGGAGAGAAGAGAGAGUGAGAGUGUGGAAAAAAAAAAAAAAAGAAUGACCUGGCCCCUCACCCUCUGCCCUCCCCGCUGCUCCUCAUAGACAG GCUGACCAGCUUGUCACCUAACCUGCUUUUGUGGCUCGGGUUUGGCUCGGGACUUCAAAAUCAGUGAUGGGAAAAAGUAAAUUUCAUCUUUCCAAAUUGAUUUGUGGGCUAGUAAUAAAA UAUUUUUAAGGAAGGAAAAAAAAAACACGUAAAACCAUGGCCAAACAAAACCCAACAUUUCCUUGGCAAUUGUUAUUGACCCCGCCCCCCCCUCUGAGUUUUAGAGGGUGAAGGAGGCUU UGGAUAGAGGCUGCUUCUGGGGAUUGGCUGAGGGACUAGGGCAACUAAUUGCCCACAGCCCCAUCUUAGGGGCAUCAGGGACAGCGGCAGACAUGAAAGACUUGGGACUUGGUGUGUUUG UGGAGCCGUAAGGCGUAUGUUAACUUUGUGUGGGUUUGAGGGAGGACUGUGAUAGUGAAGGCUGAGAGAUGGGUGGGCUGGGAGUCAGAGGAGAGAGGUGAGGAAGACAGGUUGGGAGAG GGGGCAUUGCGUCCUUGCCAAGGAGCUUGGGAAGCACAGGUAGCCCUGGCUGCAGCAGUCUUAGCUAGCACAGAUGCCUGCCUGAGAAAGCACAGUGGGGUACAGUGGGUGUGUGUGCCC CUUCUGAAGGGCAGCCCAUGGGAGAAGGGGUAUUGGGCAGAAGGAAGGUAGGCCCCAGAAGGUGGCACCUUGUAGAUUGGUUCUCUGAAGGCUGACCUUGCCAUCCCAGGGCACUGCUCC CACCCUCCAGGAGGAGGUCUGAGCUGAGGAGCUUCCUUUUCGAUCUCACAGGAAAACCUGUGUUACUGAGUUCUGAAGUUUGGAACUACAGCCAUGAUUUUGGCCACCAUACAGACCUGG GACUUUAGGGCUAACCAGUUCUUUGUAAGGACUUGUGCCUCUUGCGGGAACAUCUGCCUGUUCUCAAGCCUGGUCCUCUGGCACUUCUGCAGUGGUGAGGGAUGGGGGUGGUAUUCUGGG AUGUGGGUCCCAGGCCUCCCAUCCCUCGCACAGCCACUGUAUCCCCUCUACCUGUCCUAUCAUGCCCACGUCUGCCACGAGAGCCAGUCACUGCCGUCCGUACAUCACGUCUCACCGUCC UGAGUGCCCAGCCUCCCAAGCCCAAUCCCUGGACCCCUGGGUAGUUAUGGCCAAUCUGCUCUACACUAGGGGUUGGAGUCCAGGGAAGGCAAAGAUUUGGGCCUUGGUCUCUAGUCCUAC GUUGCCAGAAUCCAACCAGUGUGCCUCCCACAAGGAACCUUACAACCUUGUUUGGUUUGCUCCAUCAGGCGUUUGGCGCCAUCGUGGAUGGAGUCCGUGUGUGCCUGGAGAUUACCCUGG ACACCUCUGCThis cis-acting signal of tau was compared base by base with the other afore mentioned mRNAs using simple alignment. We also made comparisons with another sequence that is specific for the localization of BC1 mRNA in dendrites, the Dendritic Target Element (DTE) .In the β-actin mRNA of chicken a cis-acting signal "zipcode" has been described; a zipcode binding protein binds to this sequence and this is a prerequisite for the localization of the mRNA. The sequence is a tandem repeat of an ACACCCACACCC motif. The mRNA of β-actin has been located in the axon of the neuron and in dendritic spines ,14. β-acCofilin is a cytoskeleton modulating protein; it is also known as actin depolymerizing factor (ADF). The potential role of cofilin is to modulate the changes of actin organization that accompany neurite initiation, axonogenesis and growth cone guidance . The posVimentin has been located by RT-PCR in the axons of dorsal root ganglia (DRG) neurons. A possible sequence signal in vimentin mRNA shares some U with tau but also contains more purines, which might indicate that the protein is not exclusive to the axonal region .Bip is a protein that binds to the immunoglobin heavy chains in pre-β cells. Its mRNA shares some U with the tau sequence; nevertheless, its sequence suggests that this mRNa, like vimentin, is probably not exclusive to the axon .The heat shock proteins and grp75 messengers have similarities with the tau sequence, but once again they are probably not exclusive to the axon. They could interact with other proteins in different parts of the cell.Synuclein is a soluble unfolded protein that can aggregate into insoluble fibrils under several pathological conditions including Parkinson's and Alzheimer's diseases . The posThese analyses carried out by alignment allowed us to show that the cis-acting signals of the mRNAs examined have some homology with that of the tau messenger.The highest homology scores are:The secondary structures of these four mRNAs, which showed the closest homologies to the tau sequence, were analyzed using the program Mfold Figs. , 2, 3, 4The first messenger to be analyzed in the 3'-UTR with respect to its localization/stabilization was tau ,10. The Glucose regulated protein 75 (GRP75) is an important molecular chaperon belonging to the heat shock protein (HSP) family. It is highly expressed in conditions of glucose deprivation of glucose. Its messenger was located in the axon and it has a U-rich region. It might not be confined exclusively to the axon because this protein responds to a metabolic stress .Alfa-synuclein is involved in neurodegenerative diseases and its presence has been observed in the pre-synaptic and nuclear compartments, though the location in the nucleus has not been well documented. The synuclein messenger possesses a U-rich region; nevertheless a C interrupts the potential signal sequence. When it is wrongly folded, this protein may aggregate in the cell forming fibrils, typical of Alzheimer's and Parkinson's diseases. The aggregation of synuclein is similar to tau in Alzheimer patients, which could indicate similar intracellular behavior by both proteins .The 3'-UTR of the cofilin messenger has a U-rich region very similar to the signal sequence of tau, which on the face of it suggests that it might be located exclusively in the axon. Nevertheless, recent studies demonstrate that it participates in the shrinkage of dendritic spines associated with the long-term depression of hippocampal synapses, suggesting that it is also found in dendrites. Moreover, it is involved in neuronal development, axogenesis, guidance of the growth cone and dendrite formation. Although the cofilin messenger is present in axons, the possible participation of the protein in events related to the unplugging of synapses because of its association with actin further suggests that it is not confined to the axon but also occurs in the dendrites .The 3'-UTR of the β-actin messenger is very short and shows low homology when aligned with the tau cis-acting signal. However, when it was aligned with the dendritic target element, it showed better homology. The β-actin messenger was shown to possess a zipcode that leads it towards the dendrites instead of the axon .Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative conditions, including Parkinson's and Huntington's diseases, which are characterized by accumulation of protein aggregates . These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones, suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimer's disease, a neurodegenerative disorder characterized by β-amyloid and tau protein aggregates. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. A direct association between the chaperones and tau protein has been demonstrated. Many results suggest that the up-regulation of molecular chaperones may suppress the formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . When weOn the basis of the results we suggested a model for mRNA localization in the axon Fig. .The mRNAs containing the U-rich region could be complexed with a protein responsible for transport toward the axon, just as HuD complexes with and stabilizes the tau messenger . HuD itsThe mechanisms determining whether a messenger such as β-actin is transported preferentially to the axon or the dendrites are poorly understood. The existence of two potentially conflicting location signals in the 3'-UTR  raises questions about how the final destination of such mRNAs is determined within the neuron.In the 3'-UTRs of some mRNAs in neurons there are cis-acting signals that direct mRNAs such as tau and GAP-43 to the axon. In general, these signals are rich in uridine and do not contain guanidine. Comparison of the Dendritic Target Element (DTE) with the 3'-UTRs of several axon-located messengers showed some homology in a specific region of the 3'-UTR. Most of the 3'-UTRs studied possess homologies with the signals involved in the localization of mRNAs in axons and dendrites. This might explain why as much β-actin is present in dendrites as in axons, though the distribution mechanisms in such cases are not understood. In addition, we found a DTE homology in the 3'-UTR of HSP70 and 90. The significance of this is not clear; some messengers are transported towards the axon or towards the dendrites as required.A sequence homologous to DTE in tau occurs near the end of the 3'-UTR, next to the polyadenylation site, which indicates that only the axon signal sequence  is functional, because mRNA degradation starts at the poly(A) site. The 3'-UTR of MAP2  possesseVery U-rich sequences in the 3'-UTR might be signals that direct some mRNAs exclusively to the axon. If we understand which signals/sequences the neuronal cell uses for the correct location of its mRNAs, it might become possible to determine which factors lead to mislocalization of messengers and of proteins, as has recently been suggested in relation to certain neurodegenerative diseases such as Alzheimer's.Rattus norvegicus genome and were located using the following GeneBank accession numbers. β-actin; NM_031144, tropomyosin 3 (Tpm3); NM_057208, cofilin; NM_017147, vimentin; NM_031140, immunoglobulin heavy chain biding protein (Bip); M14050, heat shock protein 60 (HSP60); X53585, heat shock protein 70 (HSP70); L16764, heat shock protein 90 (HSP90); S45392, glucose regulated protein (grp75); s78556, synuclein; NM_031688; NM_057114 and NM_053576 and tau; X79321. The 3'-UTRs of the mRNAs were analyzed using the program ClustalW [All the mRNAs analyzed in this study belong to the ClustalW , and theClustalW .The author(s) declare that they have no competing interests.