The kinase BsfK specifically catalyzes the phosphorylation of the precursor peptide BsfA on the Ser3 residue. BsfB1 performs dual functions to accelerate the post-translational phosphorylation and assist BsfB2 in leader peptide removal. Most importantly, the penultimate residue of leader peptide is an isoleucine rather than the conserved threonine and this isoleucine has a marked impact on the phosphorylation of Ser3 as well as leader peptide removal, implying that BsfB1 and BsfB2 exhibit a new substrate selectivity for leader peptide binding and excision. This is the first experimentally validated penultimate isoleucine residue in a lasso peptide precursor to our knowledge. In silico analysis reveals that the leader peptide Ile/Val(-2) residue is rare but not uncommon in phosphorylated lasso peptides, as this residue is also discovered in Acidobacteriaceae and Sphingomonadales in addition to Bradymonadales.Lasso peptides are ribosomally synthesized peptides that undergo post-translational modifications including leader peptide removal by B (or the segregated B1 and B2) proteins and core peptide macrolactamization by C proteins to form a unique lariat topology. A conserved threonine residue at the penultimate position of leader peptide is hitherto found in lasso peptide precursors and shown to be a critical recognition element for effective enzymatic processing. We identified a lasso peptide biosynthetic gene cluster ( C-terminal tail threads through the macrolactam ring formed by the N-terminal amino group and the carboxylic acid side chain of an aspartate or a glutamate located at position 7–9 of the core peptide via an isopeptide bond (D), which is responsible for excretion and self-resistance. B genes from actinobacteria and firmicutes are usually split into two genes: B1 encodes the N-terminal RiPP precursor recognition element (RRE) domain with homology to PqqD family proteins, while B2 encodes the C-terminal protease domain that cleaves the leader peptide of the precursor, after which the remanent core peptide is macrolactamized to form the mature lasso peptide  with a characteristic lariat topology, in which the ide bond . The numide bond . Generalide bond . Many la peptide .Beyond the class-defining leader peptide removal and core peptide macrolactamization, nine unique post-translational modifications (PTMs) are reported in total in the biosynthesis of lasso peptides up to now , among wBradymonas sediminis FA350 is a Gram-negative, rod-shaped, and facultatively prey-dependent bacterium isolated from coastal sediments in Weihai, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belongs to a novel bacterial order Bradymonadales and a novel family Bradymonadaceae in the class Deltaproteobacteria (B. sediminis FA350 (accession No. NZ_CP030032.1) and subsequent search for natural products BGCs allowed the identification of a lasso peptide BGC, which we subsequently named bsf (bsfA), a discrete RRE (bsfB1), a leader peptidase (bsfB2), a lasso peptide cyclase (bsfC) and an ABC-transporter (bsfD), two additional genes encoding a putative kinase (bsfK) and a predicted nucleotidyltransferase (bsfN) have been found in this BGC as well. Herein, we characterized the activities of BsfK, BsfB1, and BsfB2.bacteria . Sequencamed bsf . The canApexHF HS DNA polymerase for high-fidelity amplification from Accurate Biology Co. Ltd. (China). Chemical compounds and reagents were purchased from Bide Pharmatech Ltd. (China), Macklin Biochemical Technology Co., Ltd. (China) and J&K Scientific Ltd. (China) unless stated otherwise. Gene synthesis and codon optimization were performed at GENEWIZ, Inc. (China). Primer synthesis and DNA sequencing were performed at Shanghai Sangon Biotech Co. Ltd. (China). Primers used in this study are summarized in Biochemicals and media were purchased from Sinopharm Chemical Reagent Co. Ltd. (China) or Oxoid Ltd. (United Kingdom) unless stated otherwise. Enzymes were purchased from New England Biolabs Ltd. (United Kingdom) except 2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid) with a flow rate of 0.3 mL/min over a 25 min period as follows: T = 0 min, 5% B; T = 3 min, 5% B; T = 18 min, 95% B; T = 22 min, 95% B; T = 23 min, 5% B; and T = 25 min, 5% B (mAU at 220 nm). ESI-MS was performed on a Bruker AmaZon SL Ion Trap LC/MS spectrometer , and the data were analyzed using Bruker Daltonics DataAnalysis. ESI-HRMS analysis was carried out on a Bruker High Resolution Q-TOF mass spectrometry  and the data were analyzed using Bruker Daltonics DataAnalysis. Tandem MS analysis was performed by collision-induced dissociation (CID) using He as collision gas.HPLC analysis was carried out on a Thermo Fisher Dionex UltiMate 3,000 UHPLC system  using an Acclaim TM RSLC 120 C18 column  by gradient elution of solvent A  and purified by ethanol precipitation. The digested genomic DNA, p15A vector with homologous arms, T4 polymerase, and buffer were mixed and reacted in a thermocycler with the following program: 25°C, 60 min; 75°C, 20 min; 50°C, 60 min. The mixture was then electroporated into the l-arabinose induced E. coli GB05-dir competent cells and plated on appropriate Luria-Bertani (LB)-agar plates to incubate at 37°C overnight. The bsf region (approximate 22.67 kb) was cloned into the corresponding p15A-cm vectors in E. coli, and several constitutive promotors such as PTn5-Km, Papra, or the inducible promoter Ptet were subsequently inserted upstream to bsfC, respectively, leading to the constructions of p15A-cm-Tn5-km-bsf, p15A-cm-Papra-bsf, and p15A-cm-Ptet-km-bsf. Then, the plasmids were electroporated into the heterologous hosts E. coli BL21(DE3) or E. coli GB2005. In order to heterologous express the BGC in Burkholderiales hosts, the chloramphenicol resistance gene was then replaced with phiC31-site-specific integrase by recombineering, resulting the construction of p15A-phiC31-amp-Papra-bsf. This plasmid was then electroporated into Schlegelella brevitalea DSM 7029 or introduced into the specific chromosomal site of Burkholderia gladioli ATCC10248 and Burkholderia thailandensis E264 with the help of donor strain E. coli WM3064, which is auxotrophic for diaminopimelic acid , inserted into the BamHI-HindIII site of the plasmid pRSFDuet-1ATGGAAATGAAAAAAGCTAAAAACAAAATCTCTCGTAAACTGATCTACAAAAAACCGCAGCTGACCTACCACTCTACCCTGGCTGCTATCATCCGTGGTACCTCTGGTAACGAAGTTGACGGTTCTACCCCGGCTGACCGTGGTAACAACCCGGGTGACTAAbsfB1 (accession No. WP_162687378.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGCGTGAAACCCCGATGACCGAAAACCTGTTCCCGCGTGACGCTGTTTTCTCTATCCGTGAAGACCTGGTTGTTGAACAGGTTGACGACGAATTCCTGGTTCTGGACCTGCGTGGTAACGAATACTTCGGTCTGAACGCTGTTGCTCGTCACATCTGGGCTGCTATCGACGCTGGTGACTCTCTGGCTGCTATCGCTGACTCTGTTTGCGAACGTTTCGAAGTTGAACGTGAACGTGCTGCTACCGACGTTGCTGACTTCATCGCTAACCTGCTGGAACAGCGTCTGGTTTCTCGTGTTGACGCTTAAbsfB2 (accession No. WP_111331306.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGAAACCGATCCACCCGATCGCTCAGGCTGAAATGCTGGTTGAAGTTGCTATCGCTCGTGTTCTGTCTGAATTCCTGTCTATCGAAGACCTGCTGCGTCTGGTTGGTGAAGCTGCTCGTATCGCTCAGCAGTGGCCGGCTCGTTCTTGCGTTGCTTCTCCGTCTGCTGAAGCTATCAACGAAACCGCTGCTCTGTCTGAACTGCTGGCTCACCGTGCTTCTCGTCTGGTTCCGGGTGCTCAGTGCCTGCAGCGTGCTCTGGCTGGTCGTGTTTGGCTGGCTCGTCGTGGTATCGCTTCTGAAATCGTTGTTGGTTTCCGTAAACGTGGTGTTCTGGAAGGTCACGCTTGGCTGGAAGTTATGTCTCCGGACGGTCTGATCGAACTGTTCAACAACGCTGACGACGGTTACCGTGAATCTTTCCGTGAAGTTGCTGCTTAAbsfK (accession No. WP_204355063.1), inserted into the NdeI-XhoI site of the plasmid pET28aATGGAAAACACCGACCGTCTGGTTCGTTACCGTGCTTTCGGTCGTGACATCGACGGTCCGGCTGGTCTGTCTCTGTCTCCGGCTCCGGCTGAAGCTGCTCCGGGTGAAGTTCCGGTTCTGCGTCTGCAGCCGGACGCTTCTCTGCGTGTTTTCATCGACGAAAACCTGCCGCCGCTGCTGCACAACGTTGAAGACTACCCGGGTGGTCCGAAATTCTACGTTTGGCAGGAAGGTGAAGCTGTTGGTGTTCAGTACGACCGTTGGCGTACCCGTCTGATCCCGGGTGAAGGTCGTATCGACTTCGCTGAACTGCCGCCGTCTGGTCCGAAACGTGTTGAAGACGCTGGTGACGAATACGGTCGTTTCCGTTTCTCTCTGGCTATGGAACGTGTTTTCCTGCCGCTGTACGCTCTGTTCTCTATGCCGGACGCTGTTGCTCTGCACGGTTCTGCTGTTGTTCTGAACGGTGAAGCTTTCCTGTTCATCGGTCGTTCTGGTGCTGGTAAATCTACCACCGCTTACGAATTCGTTCGTCGTGGTGCTACCCTGCTGGCTGACGACCTGATCGTTGCTGACGTTGCTCGTGGTATCGCTCTGGGTGGTGCTCCGACCCTGCGTCTGTGGAAAGGTGAAGGTGCTCTGCCGGAAGCTCAGGAAGACCGTTCTCTGTGGCGTCACGACGCTTCTAAACGTTGGTTCCGTATCCCGGCTGAACGTGGTGCTGCTTCTGCTGTTCCGATCGCTGCTATCGTTATGCTGGACCCGGACACCCTGGGTGGTCAGCGTGACGTTCTGCCGGGTCTGGAAACCTCTCCGCAGCGTAAAGCTCTGACCGACCTGCTGGGTCAGACCTTCGACCTGTCTCACGGTACCCCGGAATGGATGGTTGCTCGTTTCCGTAACACCGCTCGTCTGATCCGTGAATACCCGTTCTACCACTTCCGTTACGTTAAATCTGCTGACGGTAAACCGACCCACATGGACGCTCTGTACCAGGCTATCGTTGGTCTGGCTTCTAAATAAbsfK was amplified by PCR with pET28a + bsfK as the template. After purification by agarose gel electrophoresis and restrictive digestion, the bsfK fragment was inserted into the NdeI-XhoI site of the plasmid pRSFDuet-1 + bsfA to yield the recombinant plasmid pRSFDuet-1 + bsfA + bsfK.The DNA fragment containing bsfB1 or bsfB2 was amplified by PCR, respectively. The fragments were inserted into the NcoI-HindIII site of pACYCDuet-1 and the NdeI-XhoI site of pCold-TF to yield the recombinant plasmids pACYCDuet-1 + bsfB1 and pCold-TF + bsfB2, respectively.The DNA fragment containing bsfA was transferred into E. coli BL21 (DE3) for expression. BsfA, fused to an N-terminal 6 × His tag was expressed at 20°C for 24 h with 100 μM isopropyl-β-d-thiogalactopyranoside  induction and shaking at 200 rpm. Cells were harvested by centrifugation at 4°C and re-suspended in buffer A containing 6 M guanidine hydrochloride, 20 mM NaH2PO4 (pH 7.5), 500 mM NaCl, 0.5 mM imidazole. After disruption by a low-temperature, ultra-high-pressure homogenizer, the insoluble material was removed by centrifugation at 20,000 g and 4°C for 1 h. The soluble fraction was subjected to purification using a Ni Bestarose FF column . Briefly, the column was equilibrated with at least 2 column volumes of binding buffer. Then, the pre-treated sample was loaded onto the Ni Bestarose FF column, and washed with elution buffer containing a low concentration of imidazole (25 mM) to remove the impurities. The sample was further eluted with elution buffer containing a high concentration of imidazole (1 M) to obtain the purified BsfA peptide. The eluted fractions were desalted by reversed phase (RP) HPLC on a Shimadzu LC-20AT system  equipped with a ReproSil 300 C4 column . The solvent used for RP-HPLC separation was deionized water with 0.1% trifluoroacetic acid , and acetonitrile with 0.1% TFA (solvent B). A gradient was applied: 5% B for 15 min, ramp up to 100% B over 50 min, hold at 100% B for 7 min, and ramp to 5% B over 5 min. Eluted fractions were monitored by UV absorbance at 220 nm. Desalted precursor peptides were lyophilized and stored at −20°C. The peptide was validated by HPLC-ESI-(HR)MS analysis on an Acclaim TM RSLC 120 C18 column  by gradient elution of solvent A (H2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid) with a flow rate of 0.3 mL/min over a 25 min period as follows: T = 0 min, 5% B; T = 3 min, 5% B; T = 18 min, 95% B; T = 22 min, 95% B; T = 23 min, 5% B; and T = 25 min, 5% B (mAU at 220 nm).The plasmid pRSFDuet-1 + bsfA + bsfK was electroporated solely or co-electroporated with pACYCDuet-1 + bsfB1  into E. coli BL21 (DE3) for expression. The precursor peptides were purified, desalted, lyophilized, and validated according to the procedures described above for unmodified BsfA precursor peptide.The plasmid pRSFDuet-1 + bsfK was transferred into E. coli BL21(DE3) and expressed according to the procedures described above for BsfA. Cells were harvested by centrifugation at 4°C and re-suspended in lysis buffer containing 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 5 mM imidazole and 10% (v/v) glycerol. After disruption by a low-temperature, ultra-high-pressure homogenizer, the insoluble material was removed by centrifugation at 20,000 g and 4°C for 1 h. The soluble fraction was subjected to purification using a Ni Bestarose FF column. The column was equilibrated with at least 2 column volumes of binding buffer. Then, the pre-treated sample was loaded onto the Ni Bestarose FF column, and washed with binding buffer. The sample was further eluted with elution buffer using a stepwise gradient containing different concentrations of imidazole (10–500 mM). The elution fractions were determined by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the fractions containing the recombinant protein were desalted using a PD-10 Desalting Column  into storage buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 10% (v/v) glycerol, and 1 mM DTT. The resulting protein was concentrated and stored at −80°C. The purity of the protein was determined by SDS-PAGE analysis, and the concentration was determined by the Bradford assay using bovine serum albumin (BSA) as the standard.The plasmid pET28a + bsfB1 and pCold-TF + bsfB2 were transferred into E. coli BL21 (DE3) for expression, respectively. The expression temperature was 15°C for TF-BsfB2 instead of 20°C for BsfK and BsfB1. The proteins were purified to homogeneity, and concentrated according to the procedures described above for BsfK.The plasmids pET28a + bsfA or pRSFDuet-1 + bsfA + bsfK as the template. Then, 1 μL of DpnI enzyme was added to the PCR system and incubated at 37°C for 3 h to remove the template. And the PCR system was electroporated into E. coli BL21 (DE3). After sequencing to validate the fidelity, the resulting precursor peptide variants were expressed individually, or co-expressed with BsfK and BsfB1 in E. coli BL21 (DE3), and purified to homogeneity, desalted, and lyophilized according to the procedures described above for the native precursor peptides.Plasmids containing site-directed mutations were generated by PCR amplification with pRSFDuet-1 + bsfK as the template. Then, 1 μL of DpnI enzyme was added to the PCR system and incubated at 37°C for 3 h to remove the template. The PCR system was electroporated into E. coli BL21 (DE3). After sequencing to validate the fidelity, the resulting BsfK variants were expressed in E. coli BL21 (DE3), purified to homogeneity and desalted according to the procedures described above for the native protein.Plasmids containing site-directed mutations were generated by PCR amplification with pET28a + 4HCO3 buffer (pH 7.8) to a concentration of 500 μM. 20 μg trypsin  was dissolved in 20 μL resuspension buffer (50 mM acetic acid) and heated at 30°C for 15 min before utilization for maximum activity. 10 μL resuspended trypsin was added to a total volume of 50 μL BsfA reaction system, followed by incubation at 37°C for 2 h. The reaction was quenched by adding an equal volume of CH3OH. After removal of precipitate by centrifugation, the mixtures were then subjected to HPLC-ESI-HRMS analysis according to the procedure described above for BsfA precursor peptide.The (phosphorylated) precursor peptide BsfA (or the BsfA variants) was dissolved in 50 mM NH2, 5 mM ATP, 200 μM BsfA  in the presence of 20 μM BsfB1 and 20 μM BsfK (or the BsfK variants). ATP was added last to initiate the reaction. The assays were quenched by adding an equal volume of CH3CN. After removal of precipitate by centrifugation, the mixtures were then subjected to HPLC-ESI-(HR)MS analysis according to the procedure described above for BsfA precursor peptide.The assays for BsfK  were performed at 30°C for 5 h in 100 mM Tris–HCl buffer (pH 8.0) containing 5 mM MgCl2HPO4 buffer (pH 7.5) containing 100 mM NaCl, 2 mM MgCl2, 2 mM ATP, 1 mM DTT, 100 μM BsfA in the presence of 20 μM BsfB1 and 20 μM TF-BsfB2. ATP was added last to initiate the reaction. After similar termination and centrifugation, the mixtures were then subjected to HPLC-ESI-(HR)MS analysis according to the procedure described above for BsfA precursor peptide.The assays for TF-BsfB2  were performed at 30°C for 12 h in 100 mM Kbsf cluster is prone to be silent under laboratory conditions, and the Bradymonas sediminis FA350 strain is difficult to genetically manipulate. Thus, heterologous expression of the bsf cluster has been tried in other hosts. Briefly, the DNA fragment containing all the bsf genes and the flanking regions was removed from the genomic DNA by restriction digestion and further connected to the vector p15A via Red/ET recombination. The obtained plasmid (p15A-bsf) was then transformed into E. coli BL21(DE3), E. coli GB2005 and Schlegelella brevitalea DSM 7029, or conjugated into Burkholderia gladioli ATCC10248 and Burkholderia thailandensis E264, respectively, for heterologous expression in different culture media. However, no mass corresponding to the predicted lasso peptide has been observed. Moreover, several constitutive promotors such as PTn5-Km, Papra and the inducible promoter Ptet have been inserted upstream to bsfC, respectively, but the mature lasso peptide has never been detected. This results in difficulty to demarcate the boundary between the leader and the core peptide in BsfA. It was reported that the conserved Tyr(-17), Pro(-14), and Leu(-12) residues in leader peptides were required for the recognition by the B1 proteins , Microvenator marinus V1718 (mmv), Lujinxingia litoralis B210 (llb), L. sediminis SEH01 (lss) and L. vulgaris TMQ2 (lvt), et al.  motif  motif , indicatocessing . Neverthpeptides .N-terminally hexaHis-tagged BsfA (BamHI + HindIII) and untagged BsfK (NdeI + XhoI). After purification, high performance liquid chromatography-electrospray ionization-high resolution mass spectrometry (HPLC-ESI-HRMS) was used for the detection of PTMs in the N-terminally tagged BsfA. Apart from the dominant unmodified BsfA , a small component with a mass increase of 79.9640 Da was also detected, which was barely visible when bsfK was omitted  with other proteins in the National Center for Biotechnology Information (NCBI) database.A BLASTP search using the BsfK protein sequence as input revealed that it was a hypothetical protein with a very low identity (<35%) with histidine containing phospho carrier protein (HPr) kinases . To vali omitted . This inNext, untagged BsfB1was introduced into the co-expression system above. In comparison with the BsfA and BsfK co-expression experiment , the monC-terminal Ser residues of the precursors , indicative of a distinct phosphorylated position from the known lasso peptides. Trypsin digestion and subsequent tandem MS analysis of phosphorylated precursor peptide pointed to the Ser3 residue as the modification site by BsfK . It was observed that while BsfA S3T was phosphorylated efficiently, no phosphorylation was observed with BsfA S3Y, surmising that BsfK is somewhat substrate tolerant . This fiN-terminally hexaHis-tagged BsfK was characterized in vitro in the presence of the BsfA-precursor, ATP and magnesium chloride. In accordance with the in vivo characterization, BsfK phosphorylated the precursor peptide in a very low yield, whereas no conversion was observed in the absence of ATP, magnesium chloride, or with heat-treated BsfK  tag , which was confirmed by MS/MS fragmentation analysis . A similar result was obtained when unphosphorylated precursor peptide was used instead of phosphorylated precursor. The mass of unphosphorylated BsfA core peptide  was detected and its identify was further verified by MS/MS analysis (Since the (TF) tag . HPLC-ES(TF) tag . The foranalysis . The latanalysis . These ranalysis , which iAcidobacteriaceae bacterium TAA 166  residue is also observed in a list of Sphingomonadales derived lasso peptide precursors I substitutions were still accepted as substrates from the processing machinery, albeit with lower turnover , 2014. I TAA 166 , and a n TAA 166 . Besidesecursors , suggestecursors . These aC-terminal serine residues were reported for ThcoK and SyanK (We were curious to know whether the leader peptide Ile(-2) residue in BsfA also plays a role in phosphorylation and leader peptide removal. This residue was targeted for replacement with other amino acids. Ile(-2) was first exchanged with the congener leucine residue in BsfA, which was then co-expressed with BsfB1 and BsfK. HPLC-ESI-HRMS analysis of the BsfA variant after purification and desalting revealed that only part of the precursor peptide was phosphorylated , comparend SyanK , whereasBradymonadales derived B1 proteins (The BsfA Ile(-2) substitution variants were furthermore utilized for the characterization of TF-BsfB2 activity. In contrast to the wild type BsfA that was cleaved almost completely, I(-2)T as well as I(-2)A exchanges completely hindered the removal of the leader peptide even in the presence of BsfB1 , 8B. In proteins . The intAla-replacements of Tyr(-17), Pro(-14), and Leu(-12) were also performed and tested for their effects on phosphorylation and leader peptide excision. The Y(-17)A and L(-12)A exchanges did not have any effect on the phosphorylation of BsfA, as the masses of unphosphorylated precursors are barely detected in HRMS analyses. Nevertheless, the P(-14)A variant precipitates immediately after addition to the BsfK assay, and no mass of the precursor is observed. Thus, double (Y(-17)A&P(-14)A) and triple (Y(-17)A&P(-14)A&L(-12)A) exchange variants of BsfA were generated. Surprisingly, neither the double nor the triple Ala substitution affects precursor phosphorylation . MoreoveAcidobacteriaceae and Sphingomonadales.Our results demonstrate that the kinase BsfK specifically catalyzes the phosphorylation of the Ser3 residue in BsfA, while BsfB1 performs dual functions to accelerate the post-translational phosphorylation and to assist BsfB2 in leader peptide removal. Moreover, it is noteworthy that the penultimate residue in the leader peptide is isoleucine rather than the conserved threonine in all other so far investigated lasso peptide precursors. This Ile(-2) residue has a profound effect on the monophosphorylation of Ser3 and leader peptide removal, both of which are decreased in Ile(-2) substitution variants. The presence of a penultimate Ile/Val residue was also observed in putative lasso peptide precursors from Paenibacillus dendritiformis C454, ThcoB2 from Thermobacillus composti KWC4, BaceB2 from Bacillus cereus VD115, PapoB2 from Paenibacillus polymyxa CR1, SyanB2 from Sphingobium yanoikuyae ATCC 51230 and PsmB2 from Bacillus pseudomycoides DSM 12442 are the only B2 proteins reported to be involved in the biosynthesis of phosphorylated lasso peptides  can be found in the article/in-silico analyses. D-SM and Z-JD assisted with data analysis and manuscript preparation. GZ and YD analyzed the data and wrote the manuscript. GZ, XB, and YZ designed the work. All authors contributed to the article and approved the submitted version.YD carried out the experiments. YD, WN, and LP performed the This work was supported by the National Natural Science Foundation of China (21907057 and 32170038), the Natural Science Foundation of Jiangsu Province, China (BK20190201), the Future Plan for Young Scholars, and the Fundamental Research Funds (2019GN032) of Shandong University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Numerous circular RNAs (circRNAs) have been recently identified in porcine tissues and cell types. Nevertheless, their significance in porcine spleen development is yet unelucidated. Herein, we reported an extensive overlook of circRNA expression profile during spleen development in Meishan pigs.CD226, MBD2, SAMD3, SIT1, SRP14, SYTL3 gene expressions via acting as miRNA sponges. Moreover, the circRNA_21767/miR-202-3p axis regulated SIT1 expression in a ceRNA manner, which is critical for the immune-based regulation of spleen development in Meishan pigs.Overall, 39,641 circRNAs were identified from 6,914 host genes. Among them, many circRNAs are up- or down-regulated at different time points of pig spleen development. Using WGCNA analysis, we revealed two essential modules for protein-coding genes and circRNAs. Subsequent correlation analysis revealed 67 circRNAs/co-expressed genes that participated in immnue-associated networks. Furthermore, a competing endogenous RNA (ceRNA) network analysis of circRNAs revealed that 12 circRNAs modulated Overall, our results demonstrated that the circRNAs were differentially expressed during different stages of porcine spleen development, meanwhile the circRNAs interacted with immune-related genes in a ceRNA-based fashion. Moreover, we presented biomedical researchers with RNAseqTools, a user-friendly and powerful software for the visualization of transcriptome profile data.The online version contains supplementary material available at 10.1186/s12864-023-09612-x. Sus scrofa) are an incredible resource for agriculture and food production, as well as biomedical research for the examination of human development, congenital diseases, and pathogen response networks [Pigs  is a newly discovered form of non-coding RNA (ncRNA) known for its enclosed configuration, generated via precursor mRNA back-splicing . In contSpleen is a secondary lymphoid organ involved in older erythrocyte elimination, iron recycle, capture and destruction of pathogens, and induction of adaptive immune responses , 18. BeiTo explore the presence of circRNAs during spleen development, we assessed circRNAs expression in the spleen tissues of Meishan pigs at various developmental stage. We prepared and sequenced ribo-depleted total RNA-seq libraries, as shown in the flow chart Fig. . Table STo characterize the circRNAs in the Meishan pig spleen tissues, we evaluated their sequences. In all, 39,641 candidate circRNAs were identified from 6,914 host genes, using ≥ 1 unique back-spliced read Fig. A. This iEmerging evidences support the presence of DE circRNAs during porcine skeletal muscle development , howeverp = 2e-08). The heatmap shows the expression pattern of red module, where the expression of genes in the red module tends to increase with time  . As depicted in Fig. 05) Fig. C. Moreov05) Fig. D. TherefBased on the above results, we identified 36 circRNAs in the red module, and 508 mRNAs in the darkturquoise module. We performed correlation analysis on the circRNAs/mRNAs co-expression  using the Shiny framework  and immunomodulation of several genes were significantly enriched. Cell adhesion molecules, which express on the cell surface, facilitate activities of several biological processes, including, immune response, inflammation, and embryogenesis [SIT1 was strongly modulated by the circRNA_21767/miR-202-3p axis. Based on a prior investigation, signaling threshold regulating transmembrane adaptor 1 (SIT1), encoded by the SIT1 gene, is a disulfide-linked homodimeric glycoprotein that interacts with the lymphocyte-specific transmembrane adaptor protein family to modulate the immunologic process [CircRNAs are known to modulate numerous physiological processes using distinct signaling networks . Althougogenesis . CD226 iogenesis . Herein, process . It is iSIT1, which may modulate immune activities. Our findings provide candidate ncRNA-based targets for future in vivo animal study on the molecular regulation of immune function within the porcine spleen. Finally, we designed a user-friendly software named RNAseqTools that is available for use for the visualization of transcriptomic data using the Shiny app (https://github.com/ChaoXu1997/RNAseqTool).In conclusion, this study presented the first reported catalog of circRNAs expression profile in the spleen tissues from Meishan pigs. We characterized numerous DE circRNAs that were relevant to the porcine spleen development. We also generated a comprehensive expression profile of various RNAs, particularly mRNAs and circRNAs, which, together, modulate a myriad of physiological processes within the Meishan pig spleen. Moreover, we identified a ceRNA network involving circRNA_21767/miR-202-3p/Overall, 24 healthy Meishan pigs were acquired from the Kunshan Conservation Ltd.  (Permission No. JS-C-05). Our examination involved 8 developmental stages, namely, 1d, 7d, 14d, 21d, 28d, 35d, 120d, and 180d after birth. All piglets were provided with the same standard diet, with housing at an environmentally regulated facility. Three piglets were sacrificed per treatment via an intravenous administration of pentobarbital sodium to minimize suffering. The spleen was immediately extracted and frozen in liquid nitrogen. Our animal care and treatment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Yangzhou University (No. SYXK(Su)2021-0026).Splenic total RNA extraction employed TRIZOL  following kit directions. RNA quality assessment employed an Agilent 2100 Bioanalyzer . Ribosomal RNA elimination from extracted RNA utilized the Ribozero™ rRNA Removal Kit . Subsequently, linear RNA was eliminated via an RNAse R kit . Lastly, sequencing libraries were generated with rRNA-free and linear RNA-free RNA using the NEBNext® Ultra™ Directional RNA Library Prep Kit .Sscrofa11.1). We uploaded the resulting RNA-seq information in the Gene Expression Omnibus (accession codes GSE228936). To construct the SAM file, we employed BWA [Sscrofa11.1 genome.Sequencing of generated libraries was done via the Illumina HiSeq 2500 platform . TopHat2 (v2.1.1) mapped the clear data against the porcine reference genome  website, and the mRNA 3’UTR sequences from BioMart  [We retrieved the miRNA sequences from the miRbase (er 2022) . To expler 2022)  predicteer 2022)  to examiWe performed the GO term and KEGG pathway –49 enricGAPDH (for mRNA and circRNA) and U6 (for miRNA) served as controls for normalization purpose, and all qRT-PCR reactions were performed three times, and the average adjusted value was used for analysis. Relative gene expressions were computed using the 2−ΔΔCt formula. All data analysis employed the unpaired 2-tailed Student’s t-tests; *p < 0.05, **p < 0.01, and data are provided as mean ± SD (standard error of mean).qPCR employed an Applied Biosystems qPCR apparatus and SYBR green master mix , as per kit protocols. The reaction mixture was composed of 5 µl SYBR Green Mixture , 1 µl cDNA template, 0.2 µl primer (each), and 3.6 µl deionised water, and the conditions were set as follows: 95 °C for 5 min, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. All employed primer sequences were summarized in Table SIT1-3’UTR or circRNA_21767 downstream of the pmirGLO Dual-Luciferase vector. Next, we plated cells into a 24-well plates, and co-transfected the cells with the WT (SIT1-WT or circRNA_21767-WT), or Mut , and miR-202-3p-mimics (NC-mimics). Finally, we detected both firefly and renilla bioluminescence using the DLR Gene Assay Kit . The firefly luciferase activity was then adjusted with the control renilla luciferase activity for a measurement of the constructed reporter luciferase activity.We inserted wild-type (WT) or mutant-type (Mut) SIT1-WT:TCTGTGCTGACGCCTCAGTGTCTCCTCAGCCACAGGAAGTAGGCAGTGGGGGAGGGGGTTAGAGCCTGAGAGGATATGTATGGGTATC.TCCTCCCAACCCCCAAACTCCCAGGTTTTCAGTCCTCCTTCCGGAGTTTAATCAGATGCTCCCCACTCCGGCTGCCTCATGGSIT1-Mut:CTCACATCGGTATTCTAGTGTCTCCTCAGCCACAGGAAGTAGGCAGTGGGGGAGGGGGTTAGAGCCTGAGAGGATATGTATGGGTATC.TCCTCCCAACCCCCAAACTCCCAGGTTTTCAGTCCTCCTTCCGGAGTTTAATCAGATGCTCCCCACTCCGGCTGCCTCATGGcircRNA_21767-WT:CCCAGAATCTTTTAGTCCTGCCCTACAGCTGCACCTCGTACATCAAGCTCCATATAATGTTCCTCCTTACCTCTCAAAGAACGAATCAAATCTTGGGGACCTCTTAT.TAGTCTTGTATTGATGGTTTTGCACTATTTACAGAccctACCTGAAcctatccttccatccatccaaaAAATGTAcircRNA_21767-Mut:TTTGGAATCTTTTAGTCCCATTCTACAGCCATGTTCTGTACATCAAGCTCCATATAATGTTCCTCCTTACCTCTCAAAGAACGAATCAAATCTTGGGGACCTCTTAT.TAGTCTTGTATTGATGGTTTTGCACTATTTACAGAccctACCTGAAcctatccttccatccatccaaaAAATGTABelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


C. elegansvulva is a polarized epithelial tube that has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis. Here we used endogenous fusions to show that the spectrin cytoskeleton is polarized in this organ, with conventional beta-spectrin (UNC-70) found only at basolateral membranes and beta heavy spectrin (SMA-1) found only at apical membranes. The sole alpha-spectrin (SPC-1) is present at both locations but requiresSMA-1for its apical localization. Thus, beta spectrins are excellent markers for vulva cell membranes and polarity.The There, spectrins serve as linkers to connect plasma membranes and transmembrane proteins to the actin cytoskeleton . They play numerous roles in membrane and cytoskeletal organization and stability, tissue mechanics, and morphogenesis The spectrin cytoskeleton lines the cytoplasmic sides of cell plasma membranes . Vertebrates have multiple α-spectrin and β-spectrin genes. Invertebrates such asDrosophila melanogasterandCaenorhabditis elegans have just one α-spectrin and two β-spectrin genes, a conventional β-spectrin and a larger or “heavy” β-spectrin (βH).C. elegansα-spectrin is encoded byspc-1, and β-spectrin and βH-spectrin are encoded byunc-70andsma-1, respectively .SPC-1functions with eitherUNC-70orSMA-1to control different processes. In general,UNC-70is widely expressed and plays key roles in neuron and muscle structure , whileSMA-1is expressed more specifically in epithelial cells and affects tissue morphogenesis . We also discovered a role forSMA-1in blastomere cytokinesis .Spectrins exist as tetramers composed of two dimers, each with an alpha (α) and beta (β) subunit . InC. elegans, immunolocalization and/or transgenic reporter studies in the embryo detectedUNC-70/β-spectrin at many sites of cell-cell contact butSMA-1/βH-spectrin only at apical membranes , suggesting that apical vs. basal partitioning of different β-spectrins is conserved; however, there has been limited analysis of spectrin polarity in larval or adult epitheliaInC. elegansvulva. The vulva is a polarized epithelial tube used for egg-laying. It has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis . Vulva anatomy is well characterized and apical vs. basal cell surfaces can be distinguished easily using simple light microscopy of live animals.Here we visualized the spectrin cytoskeleton in theSPC-1/α-spectrin andUNC-70/β-spectrin have been reported previously . We used CRISPR/Cas9 to tag the endogenousSMA-1/βH-spectrin protein with GFP (Methods) and then examined the localization patterns of all three fusions in the developing vulva. At the mid-L4 larval stage, vulva cells are organized into a series of 7 stacked rings (named vulA-vulF), surrounding a central lumen cavity .SPC-1::GFP outlined all 7 rings   . Thus, .2020) '.SMA-1/SPC-1tetramer formation, we found thatSMA-1/βH-spectrin is needed to localizeSPC-1/α-spectrin to apical membranes. In allsma-1(ru18)null mutants examined,SPC-1::GFP failed to localize to apical membranes and was found only at basolateral membranes .The similarly polarized distributions of β-spectrin and βH-spectrin inet al.1999). These rings adopt specific shapes and undergo stereotypical movements during morphogenesis. The vulva also must connect appropriately with the uterus to allow the passage of eggs, and with the sex muscles and neurons that control egg-laying . Many of these cell behaviors depend on the actin cytoskeleton and/or on interactions with apical or basal extracellular matrices . We observed a variety of vulva shape abnormalities insma-1mutants  and the panels assembled with Adobe Illustrator.SMA-1with GFP was performed by Suny Biotech. The tag is inserted at theSMA-1C-terminus, immediately preceding the stop codon, as shown below. This endogenous fusion is functional based on normal body morphology, brood size and embryonic viability of the homozygotes.CRISPR/Cas9-mediated genome editing to tag endogenous1) Wild type sequence:TAGatacctcaccacacgctgatcttcataTTATTCAAGCGTGGATCCAAACATTCAAAG*sma-1; asterisk is where the GFP was insertedBold TAG is the stop codon ofsma-1(syb4954[SMA-1::GFP])V2) Precise sequence knock-in,CTGTTCAAGCGTGGATCCAAACATTCAAAGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagCCAACACTTGTCACTACTTTCTgTTATGGTGTTCAATGCTTcTCgAGATACCCAGATCATATGAAACgGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGatacctcaccacacgctgatcttcataBold italics indicate silent mutations; underline indicates the GFP sequence.Strains used:GOU2043vab-10(cas602[VAB-10a::GFP])IGOU2936spc-1(cas815[SPC-1::GFP])Xunc-70(cas962[UNC-70::GFP)]VGOU3103sma-1(syb4954[SMA-1::GFP])VPHX4954sma-1(ru18) V;spc-1(cas815[SPC-1::GFP])XUP4241vab-10(cas602[VAB-10a::GFP]) I;sma-1(ru18)VUP4252


Insults to the developing cerebellum can cause motor, language, and social deficits. Here, we investigate whether developmental insults to different cerebellar neurons constrain the ability to acquire cerebellar-dependent behaviors. We perturb cerebellar cortical or nuclei neuron function by eliminating glutamatergic neurotransmission during development, and then we measure motor and social behaviors in early postnatal and adult mice. Altering cortical and nuclei neurons impacts postnatal motor control and social vocalizations. Normalizing neurotransmission in cortical neurons but not nuclei neurons restores social behaviors while the motor deficits remain impaired in adults. In contrast, manipulating only a subset of nuclei neurons leaves social behaviors intact but leads to early motor deficits that are restored by adulthood. Our data uncover that glutamatergic neurotransmission from cerebellar cortical and nuclei neurons differentially control the acquisition of motor and social behaviors, and that the brain can compensate for some but not all perturbations to the developing cerebellum. Cerebellar injury in early life can impair the development of motor and social behaviors. Here, the authors show that cerebellar cell types are differentially important for the acquisition of these behaviors in pups and adult mice. Preterm injuries affect the exponential phase of granule cell proliferation, although they often also alter the early development of all glutamatergic neurons in the cerebellum. The resulting defects lead to long-term changes in gray matter volume in the cerebellar cortex that are correlated to the severity of neural deficits in infants6. Cerebellar cortical injuries further impact the development and function of downstream cerebellar nuclei neurons, which serve as the predominant output from the cerebellum and link it to the rest of the motor network7. Accordingly, cerebellar defects can also impair the development and function of the cerebral cortex9. The combined injury to the cerebellum and neocortex may help explain the broad neural deficits observed in many infants that experience cerebellar injury during the perinatal period.Cerebellar injury in preterm infants is often associated with movement disorders, language impairments, and social deficits11. Intriguingly, over time, patients largely recover these impaired neural functions with only minor residual symptoms, most commonly persisting in their motor coordination13. Patients and apes with lesions localized to the cerebellar nuclei mainly demonstrate motor symptoms with limited recovery following the injury15. When the cerebellar cortex is the primary site of the lesion, deficits in social cognition, language, and motor performance arise, but they often persist following the initial injury with limited recovery, especially in the non-motor domains affecting cognition, sociability, language, and affect2. Similarly, studies in rodents have provided compelling evidence that disrupting cerebellar cortical function during development can lead to motor impairments, altered vocalizations, and social deficits21.Accumulating evidence suggests that the site of injury within the developing cerebellum may determine behavioral outcomes. When damage is confined to the cerebellar nuclei neuron axons that project and travel through the superior cerebellar peduncle, affected patients  can develop posterior fossa syndrome, which is hallmarked by ataxia, mutism, and changes in social interactionsThese clinical outcomes illustrate that while damage to the cerebellar cortex or to the downstream cerebellar nuclei neurons during infancy is sufficient to impair motor function, language, and social behavior, there are instances when the cerebellum can remarkably overcome perturbations and restore functions. Importantly, the degree of compensation may be linked to the cerebellar neurons that are primarily affected by the lesion, suggesting a unique role for each cerebellar neural subtype in the regulation of cerebellar-associated behaviors. These studies have inspired the need for a deeper examination of how the relatively few neuron types in the cerebellum contribute to a wide range of motor and non-motor functions. However, it remains largely unknown where in the circuit cerebellar-associated behaviors originate, whether the same neuronal subtypes contribute equally to the acquisition of these diverse behaviors, and whether the perturbation of all neuronal subtypes can be equally compensated for during development. Additionally, it remains specifically unexplored how the cerebellar nuclei contribute to the acquisition of different behaviors during postnatal life.22. The unique identities of the lineages can be used to manipulate GABAergic or glutamatergic cerebellar neurons. The Ptf1a lineage that originates in the ventricular zone gives rise to GABAergic neurons, including GABAergic cerebellar nuclei neurons and Purkinje cells23. In contrast, the Atoh1 lineage is derived from the rhombic lip and gives rise to glutamatergic neurons, including glutamatergic granule cells and cerebellar nuclei neurons  to eliminate the transport of glutamate into the synaptic vesicles of Atoh1 lineage neurons. This manipulation results in an effective silencing of fast neurotransmission in the genetically targeted neurons  from the Atoh1 lineage, which resulted in a lack of VGluT2 protein in pre-synaptic vesicles of the Atoh1 lineage, Vglut2-expressing neurons33. As a result, when an action potential arrives at the synapse, synaptic vesicles fuse to the pre-synaptic membrane but do not functionally affect the postsynaptic cells because no neurotransmitter is released into the synaptic cleft , and therefore the effects of our genetic manipulation do not actively and directly continue through adulthood. Unlike Atoh1 null and cerebellum-specific Atoh1 conditional knockout mice32, Atoh1Cre/+;Vglut2fl/fl mice were viable and survived into adulthood, likely because Atoh1Cre/+;Vglut2fl/fl mice had normal respiratory rhythms , but no Vglut2 expression was found in the granule cell layer   assays in adult animals showed that nearly all cerebellar nuclei neurons from the yer Fig. . We vali01) Fig. . Immunohtex Fig. . We visuice Fig.  since, aAtoh1Cre/+;Vglut2fl/fl conditional knockout mice have no major differences in zonal stripe patterning of VGluT2+ mossy fiber synapses in the anterior or posterior zones of the cerebellum, regions into which the spinocerebellar projections are targeted  and regularity (CV2). In line with our hypothesis, we found that loss of Vglut2 from the Atoh1 lineage resulted in a reduction of simple spike firing rate  to the prelimbic area in the medial prefrontal cortex36. Other studies have also suggested that these long-range projections from glutamatergic cerebellar nuclei neurons may mediate non-motor behaviors49. We therefore tested whether the Vglut2-expressing, Atoh1 lineage neurons make long-range projections to areas previously implicated in non-motor behavior. To investigate the complete population of neurons that were affected by our manipulation, we used an intersectional reporter allele (Rosafsf-lsl-tdTomato) that expresses tdTomato only after FlpO- (Atoh1FlpO/+) and Cre- (Vglut2IRES-Cre) mediated excision of two stop cassettes. In these mice, only the neurons with a history of Atoh1 and Vglut2 expression express tdTomato VPM Fig.  and the VPM Fig. . We alsoVPM Fig.  and red VPM Fig. . Furtherlei Fig. . We alsoons Fig. . We provAtoh1Cre/+;Vglut2fl/fl mice , nearly three-quarters of Vglut2+ neurons in the interposed nucleus were also YFP+ (73.7 ± 1.3%), and around a sixth of Vglut2+ neurons in the fastigial nucleus were also YFP+ (16.5 ± 1.6%). Overall, around half of all Vglut2+ cerebellar nuclei neurons were also YFP+ (52.5 ± 1.1%)  Fig. .Ntsr1Cre-expressing nuclei neurons send long range projections to areas associated with motor and non-motor behaviors. To this end, we crossed the Ntsr1Cre mice to Atoh1FlpO/+;Rosafsf-lsl-tdTomato mice to obtain mice that expressed tdTomato solely in the Ntsr1Cre expressing cerebellar nuclei neurons , may affect adult behaviors. We found that compared to control mice, adult Ntsr1Cre/+;Vglut2fl/fl mice had no deficits in ambulatory activity  and specific (only the glutamatergic transporter Vglut2 is deleted). We have used the elimination of vesicular transporters to study the contribution of specific circuit components in several previous studies. In these studies, we did not detect changes in synaptic connectivity or developmental compensation that overcome the functional elimination of neurotransmission72. Therefore, we propose that our genetic methods allow for testing how synaptic VGluT2-dependent neurotransmission only in neurons within the intersectional domain influences circuit function and mouse behavior. Our study confirms that Atoh1 lineage cerebellar nuclei neurons are necessary for the refinement of motor behavior, and we also unveil that Atoh1 granule cells contribute to social vocalizations during postnatal development.Our genetic silencing approach provides a compelling in vivo demonstration of how the function of This study provides in vivo experimental evidence showing that glutamatergic neurons in the cerebellum are critical for the acquisition of motor function and social behaviors. Specifically, the glutamatergic neurons in the cerebellar cortex and nuclei are differentially required for the early postnatal development of motor behavior and social behaviors. These data also raise the possibility that parallel signaling from the GABAergic nuclei neurons may provide some compensation for functional lesions of the glutamatergic nuclei neurons during development, leading to improvements in early motor and social deficits. By leveraging the ability to restore cerebellar-dependent behaviors after normalizing cerebellar cortical function, tapping into cerebellar neurons that communicate social and motor signals to extra-cerebellar regions may provide an ideal therapeutic target to restore motor and non-motor functions after developmental brain injury and disease.Ai14 ;73Ai32 ;74Ai65 ;74Atoh1Cre;75Atoh1FlpO (JAX: 036541);32Ntsr1Cre (MMRRC: 030648);57Vglut2IRES-Cre (JAX: 028863);76Vglut2fl (JAX: 012898)72. We used ear clippings from pre-weaned pups for genotyping and identification of transgenic alleles. Mice of both sexes were used in all experiments. We considered the day the pups were born as postnatal day 0 (P0). We did not observe gross differences in weight between control and Atoh1Cre;Vglut2fl/fl conditional knockout mice . The ages of the adult mice were between two and fourteen months old. All mice were kept under a 14 h/10 h light/dark cycle, a daily temperature between 68 and 72 F, and humidity between 30 and 70%.All mice included in the experiments for this manuscript were housed in a Level 3, AALAS-certified facility. The Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine (BCM) reviewed and approved all studies that involved mice. We used the following mice for our experiments: 29. We anesthetized mice with Avertin and tested them for effective anesthesia by toe or tail pinch. Analgesia was also provided. We then accessed the chest cavity and penetrated the heart with a small butterfly needle for perfusion. We first perfused with 1 M phosphate-buffered saline (PBS pH 7.4) to flush out blood until the liver turned clear. Next, we perfused the mouse body with 4% paraformaldehyde (PFA) to fix the tissue. After the tail and hind paws were stiff from fixation, we decapitated the mouse and dissected the brain from the skull or spinal cord from the spinal column. We post-fixed brain and spinal cord tissue in 4% PFA overnight or until the tissue was used for cryoprotection. We cryoprotected the tissue by serial sucrose gradients (15% → 30% sucrose in PBS) at 4 °C, each step until the tissue sinks. Finally, we froze the tissue in an optimal cutting temperature (OCT) solution and stored it at −80 °C. Brain sections were cut into 40 µm free-floating tissue sections, and spinal cord sections were cut into 25 µm sections on the slide. Cut sections were stored at 4 °C until it was used for immunohistochemistry (tissue was stored for a maximum of two months). Tissues from mice expressing the tdTomato allele were stored in aluminum foil at all steps to prevent photobleaching.We collected brain and spinal cord tissue for analyses as previously described29. We blocked tissue sections in 500 μL 10% normal goat or normal donkey serum in 0.1% Triton-X in PBS (PBS-T) for 2 h. We then incubated tissue sections overnight at room temperature in 500 μL blocking solution with primary antibodies at desired concentrations. After washing the tissue sections three times in PBS-T, we incubated the tissue for two hours in 500 μL PBS-T with secondary antibodies at desired concentrations. Subsequently, we incubated tissue sections with DAPI  in PBS for 10 min. After a final two washes, we mounted the sections using VECTASHIELD Vibrance® mounting medium . All mounted slides were stored at 4 °C until they were imaged. We used the following primary antibodies: guinea-pig (gp)-anti-VGluT2 , rabbit (rb)-anti-VGluT1 , rb-anti-Parvalbumin (PV) , rb-anti-Neurogranin (NG) , rb-anti-carbonic anhydrase 8 (Car8) , and sh-anti-tyrosine hydroxylase (TH) . We used the following secondary antibodies which were conjugated to an Alexa-488 fluorophore: goat-anti-gp , goat-anti-rb , or goat-anti-sh .We stained free-floating tissue sections as previously describedVglut2, Vglut1, YFP, and tdTomato expression in unfixed, fresh frozen tissue cut in 25 μm-thick coronal brain sections. Digoxigenin (DIG)-labeled mRNA antisense probes against Vglut2, Vglut1, YFP, and tdTomato were generated using an RNA DIG-labeling kit from Roche. The specific sequences of the antisense probes that we used were as follows:ISH was performed by the RNA ISH Core at Baylor College of Medicine using an automated robotic platform. ISH was used to visualize Vglut2: GGTGCTGGAGAAGAAGCAGGACAACCGAGAGACCATCGAGCTGACAGAGGACGGTAAGCCCCTGGAGGTGCCTGAGAAGAAGGCTCCGCTATGCGACTGCACGTGCTTCGGCCTGCCGCGCCGCTACATCATAGCCATCATGAGCGGCCTCGGCTTCTGCATATCCTTCGGCATCCGCTGTAACCTGGGCGTGGCCATCGTGGACATGGTCAACAACAGCACTATCCACCGCGGAGGCAAAGTTATCAAGGAGVglut1: CAGAGCCGGAGGAGATGAGCGAGGAGAAGTGTGGCTTTGTTGGCCACGACCAGCTGGCTGGCAGTGACGAAAGTGAAATGGAGGACGAGGCTGAGCCCCCAGGGGCGCCCCCCGCGCCGCCTCCGTCCTACGGGGCCACACACAGCACAGTGCAGCCTCCGAGGCCCCCGCCCCCTGTCCGGGACTACTGACCACGGGCCTCCCACTGTGGGGCAGTTTCCAGGACTTCCACTCCATACACCTCTAGCCTGAGCGGCAGTGTCGAGGAACCCCACTCCTCCCCTGCCTCAGGCTTAAGATGCAAGTCCTCCCTTGTTCCCAGTGCTGTCCGACCAGCCCTCTTTCCCTCTCAACTGCCTCCTGCGGGGGGTGAAGCTGCACACTAGCAGTTTCAAGGATACCCAGACTCCCCTGAAAGTCGTTCTCCGCTTGTTTCTGCCTGTGTGGGCTCAAATCTCCCCTTTGAGGGCTTTATTTGGAGGGACAGTTCAACCTCTTCCTCTCTTGTGGTTTTGAGGTTTCACCCCTTCCCCCAAGACCCCAGGGATTCTCAGGCTACCCCGAGATTATTCAGGTGGTCCCCTACTCAGAAGACTTCATGGTCGTCCTCTATTAGTTTCAAGGCTCGCCTAACCAATTCTACATTTTTCCAAGCTGGTTTAACCTAACCACCAATGCCGCCGTTCCCAGGACTGATTCTCACCAGCGTTTCTGAGGGAYFP: AGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGtdTomato: ATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAANissl staining was performed with the tissue sections mounted and dried overnight on the slides. The next day, the mounted sections were immersed in 100% xylene two times for five minutes each time. Subsequently, they were placed through a rehydration series of 3 immersions in 100% ethanol, followed by 95% ethanol, 70% ethanol, and tap water, with each step lasting 2 min. Afterward, the sections were immersed in cresyl violet solution for approximately two minutes. They were then dehydrated following a reversed order of the rehydration series and followed by a final immersion in xylene, with each step lasting up to one minute. Coverslips were mounted on the slides immediately after using Cytoseal XYL mounting media .We acquired photomicrographs of cerebellar sections using a Leica DM4000 B LED microscope with a DPX365FX camera or using a Zeiss Axio Imager.M2 microscope with an AxioCam MRc5 camera. We stitched the whole mount images for the intersectional lineage tracing together using the Adobe Photoshop Photomerge function. We adjusted color brightness and balance using ImageJ software, and we cropped the images to the desired size for figures using Adobe Illustrator.77. Prior to surgery, mice were anesthetized using a mixture of ketamine (80 mg/kg) and dexmedetomidine (16 mg/kg). During the surgery, the mouse was placed on a heated surgery pad and received additional isoflurane (1%) when necessary. The mouse’s head was stabilized by ear bars in a stereotaxic surgery rig. When the mouse was fully anesthetized, we removed the hair from the head and made an incision in the skin over the anterior part of the skull. Next, we used a dental drill to make a large craniotomy over the cerebellar cortex. The craniotomy spanned from around bregma −5.60 mm to −6.64 mm, and from around 0.5–3 mm lateral to bregma. Purkinje cells across several lobules were randomly sampled from this craniotomy, preventing any potential bias from local activity patterns  to acquire breath waveforms and used custom-written MATLAB (Mathworks) code to derive Tidal Volume, Respiratory Frequency, and Minute Volume parameters.We tested respiratory parameters in room air as described in our previous publication47. Mice were placed head down on a negative incline (−35°) that was covered with a sterile Poly-Lined drape. We measured the time until mice turned 90° in either direction. Mice were tested three times at each time point. We suspended the test if mice did not turn within 60 s or fell down the slope. We recorded the falls that occurred within 60 s.We tested the negative geotaxis reflex at P7, P9, and P11 as previously described and shown47. Mice were placed in the supine position in an empty, clean cage. We measured the time until the mice turned onto their four paws. Mice were tested three times at each time point. We suspended the test if mice did not turn within 1 min (60 s).We tested the surface righting reflex at P7, P9, and P11, as previously described and shownAtoh1Cre/+;Vglut2fl/fl mice and littermate controls, vocalizations were recorded using a CM16 microphone (Avisoft Bioacoustics). Sound was amplified and digitized using UltraSoundGate 416H at a 250 kHz sampling rate, and bit depth of 16 while Avisoft RECORDER software was used to collect the recordings. For Ntsr1Cre;Vglut2fl/fl mice and littermate controls, we measured vocalizations using a Noldus microphone and UltraVox XT software. Due to minimal congruency between the number of vocalizations recorded between these two recording systems79, we only compared mutants to control littermates whose vocalizations were measured using the same system.We measured pup ultrasonic vocalizations in a social isolation task at P7, P9, and P11. The ultrasonic vocalizations of each animal were monitored for 2 min in a sound-attenuating chamber (Med Associates Inc.). For We measured open-field activity using automated Fusion software. Mice were placed in the center of an open field (40x40x30 cm chamber) that has photo beams for detecting horizontal and vertical activity. The chamber was placed in a room with the light set to 200 lux and ambient white noise to 60 dB. Each mouse was allowed to explore the chamber for 30 min.81 to assess the sociability of adult mice. The apparatus consisted of three chambers—a center chamber with doorways to two side chambers, all of equal dimensions . For three days prior to testing, the age- and sex-matched mice to be used as partners for Atoh1Cre/+;Vglut2fl/fl, Ntsr1Cre;Vglut2fl/fl, and their control littermates during the social interaction test were placed under a wire cup for 1 h each day. The assay consisted of a 10-min habituation session followed by a 10-min test phase performed in dim lighting conditions (15 lux). During the habituation session, the test subject was placed in the center chamber and allowed to freely explore for 10 min. Once the subject returned to the center chamber, the doorways to the side chambers were blocked with plexiglass walls. Before the test phase starts, a novel partner mouse (pre-habituated to the apparatus 3 days prior) is placed under a wire cup in one side chamber, while a novel object (Lego block of similar size and color) is placed under a wire cup in the other side chamber. The plexiglass walls covering the doorways are then lifted, and the test session begins as the test subject is allowed to freely investigate all three chambers for 10 min. The amount of time spent interacting with either the novel object or novel mouse during the two phases was scored manually by an experimenter blinded to genotype during the assay using ANY-Maze. Additional activity data that was acquired from each chamber during the two sessions was calculated automatically using ANY-Maze. The placement of the novel mouse and novel object within the side chambers was randomized to prevent chamber bias.We used the three-chamber test53. As is standard for this task, mice were placed on an accelerating rod on which they locomote according to the motion of the rod (4–40 rpm over 5 min) . Time was stopped and noted when one of three events occurred; the mouse fell off, the mouse made two consecutive rotations while hanging on the rod without walking, or the mouse successfully stayed on the rod for the total duration of the trial (5 min). We recorded the trial duration for four trials per day for three consecutive days.We assessed rotarod performance using a previously established protocol51. The tremor monitor consists of a lightweight plastic container that is suspended in the air by eight elastic cords, one on each corner. The container is large enough for mice to move around and explore freely, providing us with readings related to movement. An accelerometer is mounted onto the underside of the container, where it functions to convert the detected movements into an electrical signal that is digitized using a Brownlee amplifier and records using Spike2 software. We also used the Spike2 software to detect the tremor power using a Fourier transform analysis based on two full minutes of mouse movements. For the recordings, mice were placed in the tremor monitor and were allowed to acclimate for three minutes. After this period, the recordings for analysis were initiated once the animals started actively exploring and utilizing the space.We recorded tremors using our custom-built tremor monitord for effect size (and reported these in the figure legends) by dividing the absolute difference in the parameter average of each group by the combined standard deviation.All statistical analyses were performed using MATLAB (Mathworks). When performing a two-way (sex*genotype) ANOVA, we did not observe an effect of sex or an interaction effect between sex and genotype on pup behavior. We, therefore, combined mice from both sexes in the experiments and statistical analyses. We performed a two-tailed T-test to assess differences between control and conditional knockout mice when only a one-time point was involved. For analyses of pup behavior and rotarod performance, we performed a repeated measures ANOVA analysis with a Tukey post hoc analysis to test for differences at each time point. For the analyses of electrophysiological recordings in Purkinje cells, we used a linear mixed model analysis with genotype as a fixed variable and mouse number as a random variable. We calculated Cohen’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


RNA undergoes complex posttranscriptional processing including chemical modifications of the nucleotides. The resultant-modified nucleotides are an integral part of RNA sequences that must be considered in studying the biology of RNA and in the design of RNA therapeutics. However, the current “RNA-sequencing” methods primarily sequence complementary DNA rather than RNA itself, which means that the modifications present in RNA are not captured in the sequencing results. Emerging direct RNA-sequencing technologies, such as those offered by Oxford Nanopore, aim to address this limitation. In this study, we synthesized and used Nanopore technology to sequence RNA transcripts consisting of canonical nucleotides and 10 different modifications in various concentrations. The results show that direct RNA sequencing still has a baseline error rate of >10%, and although some modifications can be detected, many remain unidentified. Thus, there is a need to develop sequencing technologies and analysis methods that can comprehensively capture the total complexity of RNA. The RNA sequences obtained through this project are made available for benchmarking analysis methods. N6-methyladenosine (m6A), and others are complex multistep modifications, resulting in wybutosine  and a sugar-phosphate backbone. The bases and sugar of RNA undergo chemical alteration, leading to >150 modified nucleotides  methods do not sequence RNA, rather RNA is converted back to DNA and the resultant DNA is sequenced; in the reverse transcription, all modifications are lost  and the >150 modifications, it is necessary to train base-calling algorithms to establish the correspondence between the nucleotides and their corresponding current patterns. Many groups have developed methods to convert the current signals to sequence  was cloned from human fibroblast DNA into the pcDNA3.1-GFP(1–10) (Addgene cat# 70219) vector. The clone sequence was as follows:UUGAGCAUUUCAUCCGGAGUCUGGCCGCCCUGACCUUCCCCCAGCCGCCUGCAGGGGGCGCCAGAGGGCCGGAGCACGGAAAGCAGCGGAUCCUUGAUGCUGCCUUAAGUCCGGCUCAGAGGGGCGCAGCGUGGCCUGGGGUCGCUAUCUUCCCAUCCGGAACAUCUGCCCUGCUGGGGGACACUACGGGCCUUCCCUUGCCUGAGGGUAGGGUCUCAAGGUCACUUGCCCCCAGCUUGACCUGGCCGGAGUGGCUAUAGAGGACUUUGUCCCUGCAGACUGCAGCAGCAGAGAUGACACUGUCUCUGAGUGCAGAGAUGGGGGCAGGGAGCUGGGAGAGGGUUCAAGCUACUGGAACAGCUUCAGAACAACUAGGGUACUAGGAACUGCUGUGUCAGGGAGAAGGGGCUCAAGGACUCGCAGGCCUGGGAGGAGGGGCCUAGGCCAGCCAUGGGAGUUGGGUCACCUGUGUCUGAGGACUUGGUGCUGUCUGGAUUUUGCCAACCUAGGGCUGGGGUCAGCUGAUGCCCACCACGACUCCCGAGCCUCCAGGAACUGAAACCCUGUCUGCCCCCAGGGUCUGGGGAAGGAGGCUGCUGAGUAGAACCAACCCCAGGUUACCAACCCCACCUCAGCCACCCCUUGCCAGCCAAAGCAAACAGGCCCGGCCCGGCACUGGGGGUUCCUUCUCGAACCAGGAGUUCAGCCUCCCCUGACCCGCAGAAUCUUCUGAUCCCACCCGCUCCAGGAGCCAGGAAUGAGUCCCAGUCUCUCCCAGUUCUCACUGUGUGGUUUUGCCAUUCAUCUUGCUGCUGAACCACGGGUUUCUCCUCUGAAACAUCUGGGAUUUAUAACAGGGCUUAGGAAAGUGACAGCGUCUGAGCGUUCACUGUGGCCUGUCCAUUGCUAGCCCUAACAUAGGACCGCUGUGUGCCAGGGCUGUCCUCCAUGCUCAAUACACGUUAGCUUGUCACCAAACAUACCCGUGCCGCUGCUUUCCCAGUCUGAUGAGCAAAGGAACUUGAUGCUCAGAGAGGACAAGUCAUUUGCCCAAGGUCACACAGCUGGCAACUGGCAGAGCCAGGAUUCACGCCCUGGCAAUUUGACUCCAGAAUCCUAACCUUAACCCAGAAGCACGGCUUCAAGCCCCUGGAAACCACAAUACCUGUGGCAGCCAGGGGGAGGUGCUGGAAUCUCAUUUCACAUGUGGGGAGGGGGCUCCCCUGUGCUCAAGGUCACAACCAAAGAGGAAGCUGUGAUUAAAACCCAGGUCCCAUUUGCAAAGCCUCGACUUUUAGCAGGUGCAUCAUACUGUUCCCACCCCUCCCAUCCCACUUCUGUCCAGCCGCCUAGCCCCACUUUCUUUUUUUUCUUUUUUUGAGACAGUCUCCCUCUUGCUGAGGCUGGAGUGCAGUGGCGAGAUCUCGGCUCACUGUAACCUCCGCCUCCCGGGUUCAAGCGAUUCUCCUGCCUCAGCCUCCCAAGUAGCUAGGAUUACAGGCGCCCGCCACCACGCCUGGCUAACUUUUGUAUUUUUAGUAGAGAUGGGGUUUCACCAUGUUGGCCAGGCUGGUCUCAAACUCCUGACCUUAAGUGAUUCGCCCACUGUGGCCUCCCAAAGUGCUGGGAUUACAGGCGUGAGCUACCGCCCCCAGCCCCUCCCAUCCCACUUCUGUCCAGCCCCCUAGCCCUACUUUCUUUCUGGGAUCCAGGAGUCCAGAUCCCCAGCCCCCUCUCCAGAUUACAUUCAUCCAGGCACAGGAAAGGACAGGGUCAGGAAAGGAGGACUCUGGGCGGCAGCCUCCACAUUCCCCUUCCACGCUUGGCCCCCAGAAUGGAGGAGGGUGUCUGGAUUACUGGGCGAGGUGUCCUCCCUUCCUGGGGACUGUGGGGGGUGGUCAAAAGACC5)] of the 5-mers of the RNA synthesized with just the canonical nucleotides.The sequence includes 691 distinct 5-mers, about 70% 5-deoxyadenosine, 2.5 mM MgCl2, and 5 mM Tris-HCl buffer pH 8.0. The digestion mixture was incubated at 37°C for 6 h.RNA from each sample (2 µg) was hydrolyzed in a 50-µL digestion cocktail containing 8 U benzonase, 5 U calf intestinal alkaline phosphatase, 0.15 U phosphodiesterase I, 0.1 mM deferoxamine, 0.1 mM butylated hydroxytoluene, 5 ng coformycin, 50 nM internal standard [N1-methyladenosine (m1A), m/z 282 → 150, 1.2 min; m6A, m/z 282 → 150, 8.7 min; 5-hydroxymethylcytidine (hm5C), m/z 274.1 → 142.1, 0.88 min; Y, m/z 245 → 191, 0.88 min. Signal intensities for each ribonucleoside were normalized by dividing by the sum of the UV signal intensities of the 4 canonical ribonucleosides recorded with an in-line UV spectrophotometer at 260 nm.After digestion, all 9 samples were analyzed by chromatography-coupled triple-quadrupole mass spectrometry (LC-MS/MS). For each sample, 600 ng of hydrolysate was injected into each of 3 technical replicates. Using synthetic standards, HPLC retention times of RNA modifications were confirmed on a Waters Acuity BEH C18 column  coupled to an Agilent 1290 HPLC system and an Agilent 6495 triple-quadrupole mass spectrometer. The HPLC system was operated at 25°C and a flow rate of 0.3 mL/min in a gradient [Escherichia coli poly(A) polymerase, followed by Oxford Nanopore's protocol for direct RNA sequencing (SQK-RNA002). RNA samples were sequenced using an Oxford Nanopore MinION Mk1B device or a 5-cell GridION sequencer, with FLO-MIN106D flow cells . Reads were aligned to the clone sequence using minimap2 v2.17 , using tIn each sample, at each nucleotide position, we compared the RNA sequence to the corresponding sequence of the DNA sense strand, and then, we calculated the error rate as a ratio of mismatches to the total sequence reads at position (matched + mismatched). Those ratios are presented as percentages in the figures.For each sample, we also break down the errors by the 4 nucleotides in the DNA template. For each of the 4  nucleotides in the DNA, we looked for the number of mismatches in the RNA. In each sample, for each nucleotide, we determined the ratio of the number of mismatches to the total number of reads for the nucleotides and reported the ratios in percentages as A-to-X, C-to-X, G-to-X, and U-to-X where X is A, C, G, and U from Nanopore. The error rates calculated as implemented in our Python scripts are the same as those from EpiNano.To compute dwell times, reads were first resquiggled using f5c version 0.8  and principal component 1 (PC2) scores are plotted.Principal component analysis (PCA) was performed by using 7 variables  for the 45 samples . The data were normalized by using the Z-score. The PCA was calculated as the total variance explained.We also carried out a PCA with up to 5,000 randomly selected reads from each sample and the current and dwell times for the 5-mers that are most different among the modifications were used as variables. To select the variables for the PCA, for each 5-mer in our data, we performed ANOVA to identify the current signals that are most variable among the samples vs within, the top 5% of the most variable current signals were used as variables. The same analysis was used to identify the dwell times for the PCA. The variables were normalized by Z-score. The PCA was calculated as the total variance explained. For each sample, we then looked for the sites with the most modified nucleotides based on the PC1 and PC2 scores. We identified the outlier sites as those with PC scores furthest by Euclidean distance from the center of the plots; then at those locations, in each read, we looked for errors and longer dwell times to identify the most likely modified nucleotides. For the m6A, biotinylated-C, and Y samples, we only looked at the A, C, and U, respectively.Another PCA was performed for each sample with 7 variables  for each nucleotide. The data were also normalized by We synthesized a set of RNA transcripts with only canonical nucleotides and also with 10 different modified nucleotides. We then performed direct RNA sequencing using the Oxford Nanopore technology on those 2 kb RNA transcripts.O-methyladenosine (Am), 2′-O-methylcytidine (Cm), 2′-O-methylguanosine (Gm), and 2′-O-methyluridine (Um). Each of the modified nucleotides was added with its corresponding canonical nucleotide in 1:100, 1:10, 1:5, and 1:2 proportions .Some of the IVT reactions included canonical nucleotides only, others included different proportions of a modified base or sugar nucleotide and the corresponding canonical nucleotide. The modified bases consist of m1A, m6A, biotinylated cytidine, hm5C, 5-methylcytidine (m5C), Y, and the modified sugars were 2′-To verify that the modified nucleotides were incorporated into the RNA transcripts, the samples were checked by chemiluminescent assay and LC-MS/MS. The samples with biotinylated cytidine were checked by chemiluminescence using streptavidin-horseradish peroxidase. Additionally, to obtain a more accurate assessment of the modifications, 9 samples were analyzed by mass spectrometry. These include 1 sample synthesized with just canonical nucleotides and samples with m1A, m6A, hm5C, and Y at 2 concentrations . All the synthesized RNA transcripts were polyadenylated, and then sequenced using the Oxford Nanopore MinION. The base-calling was carried out using Guppy, alignment was performed with Minimap2 , and seqWe started by analyzing the 10 independently synthesized and sequenced sets of RNA with just canonical nucleotides. The samples were sequenced deeply with an average coverage of 5,563 reads  and is prone to slippage in particular when transcribing A/T homopolymers  and those from EpiNano  in the samples with and without modified bases. We found that all 6 nucleotides with modified bases have highly significantly different , the cytidines in the samples with modified cytidines  and the uridines in the Y samples. By motif analysis, we found that neighboring adenosines contributed to errors in the m6A samples but not in the m1A samples, this may allow for the 2 modifications to be distinguished. There is no such combination of features that allows us to tell apart the 3 modified cytidines. While all 6 modified bases led to significantly different dwell times in the pores, dwell time alone does not tell apart the different modifications.O-methylated sugars  at different proportions in the IVT reactions. The samples are deeply sequenced . We also carried out PCA with sequence reads to ask whether we can distinguish them. We used a similar concept in population genetic studies where individuals are grouped by populations based on their genotypes of genetic markers that have different allele frequencies across populations . We usedMaterials and Methods) and at those positions, we annotated the sequence reads with errors or longer dwell time as modified nucleotides  DNA template for IVT with 10 different modified nucleotides in a range of concentrations to generate RNA transcripts with modified bases or sugars. We also included RNA transcripts with only canonical nucleotides. The incorporation of the modified nucleotides was confirmed by chemiluminescence assay and mass spectrometry analyses. The RNAs were then sequenced on a Nanopore MinION with deep coverage, most samples having >5,000 reads. These are available for benchmarking algorithms for base-calling.We generated independently 10 sets of RNA with only canonical nucleotides for comparison with their counterparts with modified ribonucleotides. Direct RNA sequencing of the samples with only canonical nucleotides revealed that the error rate of Nanopore RNA sequencing is still >10%.Our analyses show that, while some modifications can be detected with existing algorithms such as Tombo and analysis of error patterns, we are far from sequencing RNA with all modifications. To obtain the complete sequences with all modifications, several technological advancements are needed. These include reducing the error rates to <0.1%, improving the resolution from 5-nucleotide to single-nucleotide, and developing base-callers that identify the canonical bases, known modifications, and yet-to-be discovered modifications. Given the large number of ribonucleotides per cell, even if we assume 10 pg of RNA per cell, there are >10 billion ribonucleotides in each cell, and thus, even a low error rate of 0.1% has many errors. Improvement in resolution to the single-nucleotide level will facilitate base-calling. Most probably, many modifications are transient and occur at a low level, so they are difficult to detect, and therefore, technical barriers such as poor resolution need to be improved. The biggest challenge of direct RNA sequencing is base-calling. Even if the instrument can capture current signals of a single nucleotide or uses methods such as exo-seq (RNA-sequencing technologies that provide the complete sequences with all the modifications, at affordable cost, will transform RNA biology and clinical medicine. The complete sequences will elucidate the basic aspects of RNA from splicing to stability. The sequences are crucial for the development of effective RNA-based therapeutics. The exact sequences are necessary for targeting specific RNA, replacing dysfunctional ones and in manufacturing vaccines. Development of sequencing technologies, coupled with analysis methods, is a necessary breakthrough to advance RNA biology and therapeutics.jkad200_Supplementary_DataClick here for additional data file.


Peng D, Ando K, Hußmann M, Gloger M, Skoczylas R, Mochizuki N, Betsholtz C, Fukuhara S, Schulte-Merker S, Lawson ND, Koltowska K. 2022. Proper migration of lymphatic endothelial cells requires survival and guidance cues from arterial mural cells. Published 22 March 2022This error occurred during the misaligned “copy and paste” from the primer list while drafting the paper. It did not affect anyone qPCR experiments or the results. We regret any confusion or difficulties it may have caused.In the Materials and methods session - FACS and qPCR analysis, there is a mix-up for the dll4 primersCorrected sequence:GGACAAATGCACCAGTATGCGTTTGCGCAGTCGTTAATGTOriginal sequence:CGTTCCCAGGAGCCTTTTCTTTGGGATCAGGGATGGGGATThe article has been corrected accordingly.