Caenorhabditis elegans. We show that unc-4 is not required during neurogenesis but is required in the postmitotic neurons for proper synapse patterning. In contrast, unc-37 is required in both developing and postmitotic neurons. The synaptic tiling defects of unc-4 mutants are suppressed by bar-1/β-catenin mutation, which positively regulates the expression of ceh-12/HB9. Ectopic ceh-12 expression partly underlies the synaptic tiling defects of unc-4 and unc-37 mutants. Our results reveal a novel postmitotic role of neuronal cell fate determinants in synapse pattern formation through inhibiting the canonical Wnt signaling pathway.Neuronal cell fate determinants establish the identities of neurons by controlling gene expression to regulate neuronal morphology and synaptic connectivity. However, it is not understood if neuronal cell fate determinants have postmitotic functions in synapse pattern formation. Here we identify a novel role for UNC-4 homeobox protein and its corepressor UNC-37/Groucho, in tiled synaptic patterning of the cholinergic motor neurons in  Drosophila homolog of the vertebrate Isl1, islet, specifies motor neurons and interneurons in the ventral nerve cord  homeobox proteins, respectively, function cooperatively to specify the glutamatergic mechanosensory neurons  type transcription factor, UNC-3, is required for both specification and maintenance of the cholinergic motor neuron identities , the ventrally innervating D-class (VD) GABAergic motor neurons innervate the dorsal body wall muscles instead of the ventral body wall muscles, due to the ectopic expression of the genes which are normally expressed only in the dorsally innervating D-class (DD) neurons  stage when the VA neurons are born, suggesting that this miswiring is likely due to the partial cell fate transformation of the A-type motor neurons to the B-type motor neurons  by repressing the alternative B-type neuronal fates (VBs and DBs) in  elegans . The rep subunit . While trneurons . In wildrneurons . However neurons . Tempora neurons . Howevernnection . Recent elopment , suggestunc-4 and unc-37 in precise synapse pattern formation in the DA class cholinergic motor neurons. DA neurons exhibit a unique ‘tiled’ synaptic innervation pattern, in which the synaptic domain of a DA neuron does not overlap with those from the neighboring DA neurons  system showed that unc-4 is required for synaptic tiling in the postmitotic DA neurons, but not required during the development of DA neurons. On the other hand, unc-37 is required in both the developing DA neurons and the postmitotic DA neurons. Interestingly, the expression patterns of A-type and B-type cholinergic neuronal cell fate markers are largely unaffected in the DA8 and DA9 neurons of unc-4 and unc-37 mutants, suggesting that the synaptic tiling defects in unc-4 and unc-37 are less likely due to the secondary defects of the cell fate determination. We also found that the Wnt-induced ectopic expression of ceh-12 homeobox gene underlies, at least in part, the synaptic tiling defects of unc-4 mutants. Taken together, we provide the postmitotic roles of cell fate determinants in precise synapse pattern formation.In this study, we report a novel postmitotic role for  neurons . Previou neurons . In the en passant synapses at specific subaxonal regions onto the dorsal body wall muscles (mizIs3) with improved color contrast between the two most posterior DA neurons, DA8 and DA9, under the fluorescent microscope. mizIs3 expresses the synaptic vesicle-associated protein, RAB-3, fused with a brighter GFP variant (GFPnovo2)   and ZF1 FPnovo2)  in all DFPnovo2) . As prevFPnovo2) .unc-4(e120) null mutants, we found significant overlap between the DA8 and DA9 synaptic domains: the synapses of DA8 and DA9 are intermingled in the region of overlap (unc-4 mutants (mig-13 promoter used to express ZIF-1 and mCherry::RAB-3 (see below). Indeed, the synaptic pattern of DA8 and DA9 in unc-4(e120); mizIs3 resembled that of unc-4(e120); wyIs446, which carries another synaptic tiling marker (wyIs446) we used in our previous work , which carry a single amino acid substitution (H539Y)  did not enhance the synaptic tiling defects of unc-37(e262) . As physical contact between the DA8 and DA9 axons is required for synaptic tiling (Similar to  (H539Y) , exhibitc tiling . Moreove37(e262) . This re neurons . Approxic tiling , we exclunc-4 and unc-37 function together in tiled synaptic innervation, we examined the synaptic tiling in unc-4(e26) mutants which carry a missense mutation in the eh1 domain that has been shown to disrupt the physical interaction between UNC-4 and UNC-37 (unc-4(e120) null mutants, we observed severe synaptic tiling defects between the DA8 and DA9 neurons in unc-4(e26) mutant animals  domain . To furtd UNC-37 . Similar animals . This resmp-1/Sema, plx-1/Plexin, and rap-2/Rap2A, DA8 and DA9 show severe synaptic tiling defects –Plexin signaling controls the synaptic tiling of DA8 and DA9 . In the  defects . Since U mutants . This resmp-1-dependent manner  mutants was too dim to examine its subcellular localization in a reliable manner due to the diminished itr-1 promoter activity, which we used to express PLX-1::GFP (unc-4(e2322ts)  did not rescue the synaptic tiling defects of unc-4 or unc-37 mutants (data not shown).We have previously shown that PLX-1::GFP is localized at the anterior edge of the DA9 synaptic domain where it locally inhibits synapse formation in a t manner . In unc-affected , suggestX-1::GFP . In a tee2322ts) , which sDA9 axon , suggest mutants . Howeverunc-4 and unc-37 mutants, we observed the ectopic synapse formation anterior to the PLX-1::GFP domain  under the DA neuron-specific promoter (Punc-4c) did not rescue the synaptic tiling defects of unc-4 and unc-37 mutants (data not shown). It is therefore possible that unc-4 and unc-37 regulate synaptic tiling by controlling the expression of previously uncharacterized Sema–Plexin signaling components.While PLX-1::GFP localization was not affected in P domain . This phormation . However mutants , and theunc-4 and unc-37 determine the A-type motor neuron fates by repressing the expression of B-type motor neuron-specific genes  and unc-37(e262) mutant animals, the expression of Punc-53 was not affected in both anterior and posterior DA8/9 neurons, suggesting that DA8 and DA9 retain A-type identity , Pglr-4 (otIs476), and Pitr-1 (wyIs320) . In unc-37 mutant animals, the expression of these DA9 specific markers was mostly unaffected, with a small fraction of animals (7/100) lacking Pglr-4 expression in DA9  . These DwyIs320) . In all ed above . The Pitell body . Howevern in DA9 .unc-4 and unc-37 mutants are largely unaffected, and therefore, it is unlikely that the synaptic tiling defects of unc-4 and unc-37 mutants are due to the secondary defects of cell fate transformation of the DA neurons.Together we conclude that despite the subtle changes in the expression of some of the cell fate markers, the cell fates of DA8 and DA9 in unc-4 and unc-37 function by using the AID system to conditionally control the degradation of UNC-4 and UNC-37 in a spatiotemporally controlled manner. A plant F-box protein, TIR1, mediates the degradation of AID-tagged proteins in an auxin-dependent manner (unc-4 and unc-37 with AID and blue fluorescent protein (BFP) at the 3’ end of the unc-4 locus (miz40[unc-4::AID::BFP]) and unc-37(miz36[unc-37::AID::BFP]) (unc-4(miz40) and unc-37(miz36) with a strain carrying mizSi3 (Punc-4c::TIR1), a transgene that expresses TIR1 specifically in the DA neurons under the DA neuron-specific promoter (Punc-4c) (unc-4(miz40); mizSi3 and unc-37(miz36); mizSi3 animals did not exhibit uncoordinated locomotion phenotype (data not shown) or synaptic tiling defects in the absence of auxin (unc-4(miz40); mizSi3 and unc-37(miz36); mizSi3 animals were grown in the presence of the water-soluble synthetic auxin analog (K-NAA) (unc-4(miz40); mizSi3 and unc-37(miz36); mizSi3 animals grown on K-NAA-containing plates did not exhibit backwards locomotion defects, which is attributed to the AVA>VA wiring defects in unc-4 and unc-37 mutants, suggesting UNC-4 and UNC-37 are fully functional in the VA neurons during DA-specific degradation of UNC-4 and UNC-37. Using this system, we first conducted the DA-specific continuous degradation of UNC-4 and UNC-37 by placing the L4 parental animals on K-NAA-containing plates and observing the progeny at the L4 stage. We observed severe synaptic tiling defects between the DA8 and DA9 synaptic domains (unc-4 and unc-37 are required in the DA neurons for proper synaptic tiling.We next determined in which cells t manner . Using CD::BFP]) . ConsistD::BFP])  at the LD::BFP]) . We thenPunc-4c) . unc-4 , we obse (K-NAA) . Further domains , suggestunc-4 and unc-37 cDNAs under the DA-specific unc-4c promoter in unc-4(e120) and unc-37(e262) mutant animals, respectively. We co-expressed his-24 fused to GFPnovo2, under the unc-4c promoter to determine which DA neurons carry the extrachromosomal array. We observed significant rescue of the synaptic tiling defect when unc-4 is expressed in both DA8 and DA9 neurons of unc-4 mutant animals  mutants carry a point mutation (L121F) that resides in the homeodomain (unc-4(e2322ts) mutation is permissive at 16°C and restrictive at 25°C (unc-4(e2322ts) mutants did not exhibit synaptic tiling defects compared with wild type (unc-4(e2322ts) mutants showed severe synaptic tiling defects  mutant animals from 25°C to 16°C at the L1 stage ; mizSi3 and unc-37(miz36); mizSi3 were cultured on K-NAA containing plates, and their progeny were transferred at the early L1 stage to the control plates (unc-4(e2322ts) mutants, we did not observe any synaptic tiling defects when UNC-4 was degraded during embryonic development .Next, we examined if L4 stage . Consistradation . Togethe defects . This suunc-4 and unc-37 are required for the maintenance of the synaptic tiling in the postmitotic DA neurons, we conducted postembryonic degradation of UNC-4 and UNC-37 at a later larval stage (mid-L2) and examined the synaptic phenotype at the L4 stage . Pceh-12::GFP expression was not detected in DA8 and DA9 of wild type at L1, L2, or L4 stage  in wild-type animals. We observed significant synaptic tiling defects in these animals  that function redundantly with ceh-12 under the control of unc-4 and unc-37 in synaptic tiling ; bar-1(ga80) and unc-37(e262); bar-1(ga80) double mutants. Consistent with our idea, bar-1(ga80) partially suppressed the synaptic tiling defect of unc-4 and unc-37 mutants  and ventral (U3-5) muscles ; unc-4(e120) and ceh-12(gk391) unc-37(e262) double mutants. This suggests that ceh-12 is not the only Wnt effector gene that regulates synaptic tiling and is upregulated in the unc-4 and unc-37 mutants. Future single-cell RNA sequencing of the DA8 and DA9 neurons in unc-4 mutants, as well as genetic suppressor screening of the synaptic tiling defects in the ceh-12; unc-4 and ceh-12 unc-37 mutants will be necessary to identify additional factors that function redundantly with ceh-12.UNC-4 inhibits canonical Wnt signaling to repress  neurons . Consistnections . We alsobar-1 and unc-4 (and unc-37) indicates that the canonical Wnt signaling pathway is required for the proper tiled synaptic innervation in the DA neurons. Specifically, Wnt signaling functions as a negative regulator of synaptic tiling , unc-4(e26), unc-4(e2322ts), unc-4(miz40), unc-37(e262), unc-37(miz36), plx-1(nc36), ceh-12(gk391), bar-1(ga80). Genotyping primers are listed in the supplemental material.Bristol N2 strain was used as a wild-type reference. All strains were cultured in the nematode growth medium (NGM) with OP50 as described previously . Unless mizIs3 ; wyIs320 ; mizEx362 ; mizEx365 ; mizEx410 ; hdIs1  ; wdEx419) (otIs476 (Pglr-4::TagRFP) ) ; mizEx408, mizEx409 ; mizEx434, mizEx435 ; mizEx436 ; mizEx437, mizEx439 ; mizEx440, mizEx441 .The following transgenes were used in this study: , rol-6) ; mizEx39su1006)) ; otIs476:TagRFP) ; wdIs62 -119(+)) ; mizEx39mizIs3), we used the ZIF-1/ZF1 degradation system , the pSM vector (a kind gift from S. McCarroll and C. I. Bargmann). unc-4, unc-37, ceh-12, and unc-55 cDNAs were amplified with Phusion DNA polymerase (NEB) from N2 cDNA library synthesized with Superscript III first-strand synthesis system (Thermo Fisher Scientific). The amplified cDNAs were cloned into the AscI and KpnI sites of pSM vector using Gibson assembly method  was inserted at the 3’ end of the 5’ homology arm. TIR1 from pLZ31 (Addgene #71720) was amplified and cloned into AscI and KpnI sites of the pSM plasmid containing the unc-4c promoter at the SphI and AscI sites. Punc-4c::TIR1::unc-54 utr from this plasmid was amplified and cloned into the SfrI site of the repair template by Gibson assembly.The SCI site  were ampunc-4#1:TTTGAACCGTGCCCTCCAT, unc-4#2:GCGACTAATGCATTGACTA, unc-37#1:ACTGGATCAGGGGAGAAGA, unc-37#2:CGATTATCGTACAGAATAG, and oxTi365: CGGTCGCGGTACTCCAAAT.pTK73 plasmid  was usedunc-4(miz40[unc-4::AID::BFP]), unc-37(miz36[unc-37::AID::BFP]), and mizSi3 (Punc-4c::TIR1-loxP-Pmyo-2::GFP-NeoR-loxP) were generated using a dual-selection method as previously described (unc-4(miz40), unc-4 repair template plasmid, two unc-4 sgRNA plasmids and Cas9 plasmid (Addgene #46168) (unc-37(miz36), unc-37 repair template plasmid, two unc-37 sgRNA plasmids and Cas9 plasmid were co-injected into young adults. The screening of the genome edited animals was conducted according to previous works  and screened for animals that lack Pmyo-2::GFP expression. The successful genome editing was confirmed by Sanger sequencing.CRISPR/Cas9 genome editing escribed . To gene #46168)  were co-us works . Brieflyus works . The fulmizSi3 (Punc-4c::TIR1-loxP-Pmyo-2::GFP-NeoR-loxP), we injected Punc-4c::TIR1 repair plasmid, oxTi365 gRNA, and Cas9 plasmid into wild-type animals and screened animals with uniform and penetrant Pmyo-2::GFP expression. For simplicity, we label mizSi3(Punc-4c::TIR1-loxP-Pmyo-2::GFP-NeoR-loxP) as mizSi3(Punc-4c::TIR1).For the generation of hdIs1[Punc-53::GFP] (DA-specific), mizEx396 (DB-specific), wdEx419[Pacr-16::GFP] (DB-specific), wdEx60[Pacr-5::GFP] (DB-specific), wyIs320 (DA9-specific), otIs476[Pglr-4::TagRFP] (DA9-specific), mizIs3 (DA9 specific). mCherry::RAB-3 expression in mizIs3 was used as an indicator of the mig-13 promoter activity. PLX-1::GFP signal was used to examine the itr-1 promoter activity. Scoring of the DA-, DB-, DA9 cell fate marker expressions in DA8 and DA9 neurons was conducted using ZEISS Axioplan two fluorescent microscope, except Pitr-1 in unc-4(e120) mutants, which was examined under the ZEISS LSM800 confocal microscope.The following transgenes were used as cell fate markers: unc-4(e2322ts) mutants were transferred to 25°C (restrictive temperature). Newly hatched L1 animals from the parental animals were transferred to 16°C (permissive temperature) until L4 to examine the synaptic tiling of DA8 and DA9 neurons. For postembryonic knockdown, the L4 parental animals of unc-4(e2322ts) mutants were transferred to 16°C (permissive temperature) at L4 stage. Newly hatched L1 animals were transferred to 25°C (restrictive temperature) until L4 to examine the synaptic tiling of DA8 and DA9 neurons. Animals that were not transferred were used as controls. Wild-type controls were cultured at 16°C and 25°C.For embryonic knockdown, the L4 parental animals of 2O to prepare a 400 mM stock solution. The 4 mM working solution of K-NAA was prepared by diluting a stock solution with M9 buffer. 1000 μL of 4 mM K-NAA was added to the NGM plates with OP50 bacteria and were allowed to dry overnight at room temperature. To induce protein degradation, experimental animals were transferred onto the K-NAA plates and were kept at room temperature. As a control, animals were transferred onto the control (M9) plates. For embryonic degradation of UNC-4 and UNC-37, the L4 parental unc-4(miz40);mizSi3 and unc-37(miz36);mizSi3 animals were transferred to K-NAA plates at L4 stage. Newly hatched L1 animals from the parental animals were transferred to the control (M9) plates until L2 or L4 to examine the synaptic tiling of DA8 and DA9 neurons. For postembryonic degradation of UNC-4 and UNC-37, the L4 parental unc-4(miz40);mizSi3 and unc-37(miz36);mizSi3 animals were transferred to the control (M9) plates at L4 stage. Newly hatched L1 (early postembryonic degradation) or mid-late L2 (late postembryonic degradation) animals from the parental animals were transferred to K-NAA plates until L4 to examine the synaptic tiling of DA8 and DA9 neurons.The synthetic auxin analog, α-napthaleneacetic acid (K-NAA) , was disC. elegans using a Zeiss LSM800 Airyscan confocal microscope  with oil immersion lens 63× magnification . Worms were immobilized on 2.5% agarose pad using a mixture of 7.5 mM levamisole (Sigma-Aldrich) and 0.225 M BDM  (Sigma-Aldrich). Images were analyzed with Zen software (Carl Zeiss) and ImageJ . Twenty to 26 Z-stack images were taken for each animal to encompass the cell bodies, axons, and synapses of the DA8 and DA9 neurons. The definition of DA8/DA9 synaptic overlap was defined by the distance between the most anterior DA9 synapse and the most posterior DA8 synapses  was used for statistical analyses. We applied the one-way ANOVA method with post hoc Tukey’s multiple comparison test for comparison among three or more parallel groups with multiple plotting points, and chi-square test (with Yates’ continuity corrected) for comparison between two binary data groups. Data were plotted with error bars representing standard errors of mean (SEM). *, **, and *** represent p-value < 0.05, < 0.01, and < 0.001, respectively. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:The reviewing team recognizes the importance of defining postmitotic function of transcription factor. They also found it interesting that your data showed that unc-4 and unc-37 function differently in synapse tiling. Overall, this study provided new insights in the molecular mechanisms of synapse tiling.Decision letter after peer review:C. elegans" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by Kang Shen as the Reviewing Editor and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Sustained expression of unc-4 homeobox gene and unc-37/Groucho specifies the spatial synaptic organization in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Life. One reviewer summarized your findings as below. This manuscript by Kurashina et al. describes a novel post-mitotic role in synaptic patterning for a cell fate determining gene, unc-4, and its co-repressor, unc-37. The DA neurons in C. elegans are cholinergic motor-neurons that exhibit unique synaptic tiling of their dorsal axonal segments. Mizumoto et al. has previously shown that Semaphorin-Plexin signaling is required to establish the tiling between DA8 and DA9, by functioning in cis in the DA9 neuron. Using temperature-sensitive mutant of unc-4, as well as a combination of CRISPR/Cas9 genome editing with the AID system for specific temporal degradation, the authors nicely examine the spatiotemporal requirement of unc-4, and show that unc-4 is required only post-mitotically for synapse tiling, but not during the development of the DA neurons. Interestingly, activity of the corepressor unc-37 is required both during development and postmitotically for correct tiling. unc-4 and unc-37 are suggested to function by inhibiting the canonical wnt signaling. Overall this is an interesting study that sheds light on our understanding of the post-developmental role of cell fate genes in synapse patterning.Both reviewers found your manuscript interesting and potentially suitable for publication in eThe reviewers did suggest the following essential revisions.Essential revisions:1) The authors present in their introduction the results from Kerk et al., regarding the role of unc-4 as a cell fate determining gene for the VAs and DAs. Kerk et al. have shown that UNC-4 is specifically required for the expression of DA genes, without affecting ACh pathway genes. Table 1, however, doesn't fully recapitulate the same results and actually shows that unc-4 and unc-37 mutants do not exhibit significant cell fate defects. The authors use these results to argue in the discussion that the synaptic patterning defects can occur independent of the cell fate transformation. The issue of unc-4 as a cell fate-determining gene of A type motor neurons needs to be more clearly addressed. The authors should test whether acr-5 expression is elevated in DAs in unc-4 and unc-37 mutants .2) Both reviewers had some concerns about the tiling markers. The authors use a clever strategy to assess tiling of individual cholinergic motor neurons using DA8 and DA9 as a model, but in some cases observe variable degradation of the RAB-3::GFPnovo, presumably due to weak expression of ZIF1 in some of the mutants. This makes it a little difficult to assess the tiling defects in some of the figures. The residual GFPnovo signal seems to be defined based on colocalization with the more broadly expressed mCherry::RAB-3 marker, but no data is shown for the extent of colocalization in the absence of ZIF1. This analysis would benefit from more explanation.In Figure 1, the anterior extension of the DA9 synapses in the unc-4 mutants is quite dramatic. However, all the anterior ectopic puncta shown in Figure 1C seem to co-localize with a green puncta. The authors should make sure that there is no ectopic expression of mig-13 in the DA8 neuron on unc-4 mutant. I don't have a similar concern for the unc-37 mutant because there is little perfect colocalization of the anterior synapses in the unc-37 mutants.3) Two reviewers suggested to use plx-1::GFP to provide more insight in the phenotype of unc-4. Since the itr-1::plx-1::gfp is too dim in the unc-4 mutants, plx-1::plx-1::gfp (either transgene or knock in) might provide useful insight in how unc-4 controls plx-1. Of course, it could also be that plx-1 is expressed in many cells, in that case, it would be hard to have subcellular resolution.4) In Figure 2+3, the authors drew the conclusion that unc-4 is not required during embryogenesis for synaptic connection. Since they imaged the animals during L4, it is possible that the unc-4 is required for both early and late for synaptic tiling. They should score the phenotype in late L1 or L2 to make sure that the phenotype is not there in the embryonic KD experiments.Reviewer #3 (Recommendations for the authors):Do the RAB-3::GFPnovo and RAB-3::mCherry markers always have complete overlap, for example in DA9? I'm wondering if variability in the extent of labeling across the 2 markers could influence the ability to assess tiling defects, for example in cases where the RAB-3::GFP marker is only partially degraded.The previously defined roles for unc-4/unc-37 focused around AVA connectivity with post-embryonic born VA neurons. The authors provide evidence that a subset of neuronal identity markers in DA8 and DA9 neurons are largely unchanged in unc-4 and unc-37 mutants. Do the authors envision that unc-4 and unc-37 have distinct roles in embryonic born DA neurons from those in post embryonic born VA neurons? Or perhaps additional roles?The downstream pathways  identified as important for tiling strongly overlap with those previously identified as important for neuronal fate determination. While perhaps not absolutely critical, identification of the proposed additional Wnt-regulated TF that is important (with ceh-12) for tiling might distinguish specific mechanisms for regulation of tiling and be a strong addition the story.Related, has tiling been examined in VA neurons? Might unc-4/unc-37 have roles in both neuronal fate determination and later roles in synaptic tiling in these neurons? I don't expect the authors to resolve these questions experimentally, but a more direct discussion might help to clarify. Essential revisions:1) The authors present in their introduction the results from Kerk et al., regarding the role of unc-4 as a cell fate determining gene for the VAs and DAs. Kerk et al. have shown that UNC-4 is specifically required for the expression of DA genes, without affecting ACh pathway genes. Table 1, however, doesn't fully recapitulate the same results and actually shows that unc-4 and unc-37 mutants do not exhibit significant cell fate defects. The authors use these results to argue in the discussion that the synaptic patterning defects can occur independent of the cell fate transformation. The issue of unc-4 as a cell fate-determining gene of A type motor neurons needs to be more clearly addressed. The authors should test whether acr-5 expression is elevated in DAs in unc-4 and unc-37 mutants .acr-5::GFP using the transgene used by Winnier et al., 1999 and confirmed that acr-5 is mis-expressed in many of the A-class motor neurons in the ventral nerve cord  Both reviewers had some concerns about the tiling markers. The authors use a clever strategy to assess tiling of individual cholinergic motor neurons using DA8 and DA9 as a model, but in some cases observe variable degradation of the RAB-3::GFPnovo, presumably due to weak expression of ZIF1 in some of the mutants. This makes it a little difficult to assess the tiling defects in some of the figures. The residual GFPnovo signal seems to be defined based on colocalization with the more broadly expressed mCherry::RAB-3 marker, but no data is shown for the extent of colocalization in the absence of ZIF1. This analysis would benefit from more explanation.In Figure 1, the anterior extension of the DA9 synapses in the unc-4 mutants is quite dramatic. However, all the anterior ectopic puncta shown in Figure 1C seem to co-localize with a green puncta. The authors should make sure that there is no ectopic expression of mig-13 in the DA8 neuron on unc-4 mutant. I don't have a similar concern for the unc-37 mutant because there is little perfect colocalization of the anterior synapses in the unc-37 mutants.unc-4 mutants is due to the compromised ZIF-1/ZF1 degron system in the mizIs3 synaptic tiling marker used in this study, we examined the synaptic tiling defects using another synaptic tiling marker, wyIs446, that we used in our previous work . Because wyIs446 does not use the ZIF-1/ZF1 degron system, both DA8 and DA9 express GFP::RAB-3, while mCherry::RAB-3 is only expressed in DA9. In both wildtype and unc-4 mutants, DA9 expressed both GFP::RAB-3 and mCherry::RAB-3, and GFP and mCherry puncta are nicely colocalized. In this condition we still observed drastic synaptic tiling defects in the unc-4 mutants. Therefore, we believe that the synaptic tiling defects observed in unc-4; mizIs3 is not due to the compromised ZIF-1/ZF1 degron system. We included their representative images and signal histogram of GFP and mCherry signals in Figure 1—figure supplement 1.To exclude the possibility that the synaptic tiling defects observed in unc-4 mutants. In wildtype, upon increasing the image brightness, mCherry::RAB-3 signal is detectable in the DA9 cell body but not in the DA8 cell body. In unc-4 mutants, we did not observe mCherry signal in the DA8 cell body, suggesting that DA8 does not express the DA9 marker, mCherry::RAB-3. Therefore, we believe that all of the synaptic puncta labeled with mCherry::RAB-3 are DA9 synapses. This result is included in Figure 1—figure supplement 2.We also examined the expression of mCherry::RAB-3 in the DA8 cell body of unc-4; mizIs3 is due to the misexpression of the markers used in this strain.In conclusion, we did not find any evidence that suggests the synaptic tiling defects observed in 3) Two reviewers suggested to use plx-1::GFP to provide more insight in the phenotype of unc-4. Since the itr-1::plx-1::gfp is too dim in the unc-4 mutants, plx-1::plx-1::gfp (either transgene or knock in) might provide useful insight in how unc-4 controls plx-1. Of course, it could also be that plx-1 is expressed in many cells, in that case, it would be hard to have subcellular resolution.plx-1 locus using CRISPR/Cas9 genome editing, but we did not observe reliable level of expression from the dorsal axons including DA9. The transgenic overexpression of Pplx-1::plx-1::gfp highlighted the neuronal PLX-1::GFP in DA9 judged based on its commissural localization, but the signals from the other neurons and non-neuronal cells (hypodermis and muscles) masked the subcellular localization of PLX-1::GFP at the synaptic tiling border. At least, we observe no clear reduction in the overall expression level of PLX-1::GFP in unc-4 mutants.We examined the expression and localization of the endogenous PLX-1 by inserting GFPnovo2 cDNA at the 3’ end of the endogenous unc-4 and unc-37 controls the expression of plx-1, we examined the expression of Pplx-1::gfp in unc-4 and unc-37 mutants, and found that there was slight reduction in % animals expressing Pplx-1::gfp in the anterior DA8/9 of unc-37 mutants . Given the fully penetrant synaptic tiling defects, we do not think that slight reduction in plx-1 expression is responsible for the synaptic tiling defects of unc-4 and unc-37 mutants. We also found that the expression of plx-1 cDNA under the DA neuron-specific promoter (Punc-4c) did not rescue the synaptic tiling defects of unc-4 and unc-37 mutants (not shown). Therefore, the loss or reduction of plx-1 expression is unlikely the mechanism underlying the synaptic tiling defects of unc-4 and unc-37.To test if 4) In Figure 2+3, the authors drew the conclusion that unc-4 is not required during embryogenesis for synaptic connection. Since they imaged the animals during L4, it is possible that the unc-4 is required for both early and late for synaptic tiling. They should score the phenotype in late L1 or L2 to make sure that the phenotype is not there in the embryonic KD experiments.unc-4 and unc-37 at early L2 stage. Consistent with our observation at L4 stage animals, we found that embryonic knockdown of unc-37 but not unc-4 caused synaptic tiling defects at L2 stage. This result further strengthens our conclusion that unc-4 is not required during DA neurogenesis at embryonic stage to control their synaptic tiling. The data is included in Figure 4.We examined the synaptic tiling defects of the embryonic knockdown of Reviewer #3 (Recommendations for the authors):Do the RAB-3::GFPnovo and RAB-3::mCherry markers always have complete overlap, for example in DA9? I'm wondering if variability in the extent of labeling across the 2 markers could influence the ability to assess tiling defects, for example in cases where the RAB-3::GFP marker is only partially degraded.We have answered this comment in the essential revision #2The previously defined roles for unc-4/unc-37 focused around AVA connectivity with post-embryonic born VA neurons. The authors provide evidence that a subset of neuronal identity markers in DA8 and DA9 neurons are largely unchanged in unc-4 and unc-37 mutants. Do the authors envision that unc-4 and unc-37 have distinct roles in embryonic born DA neurons from those in post embryonic born VA neurons? Or perhaps additional roles?unc-4 and unc-37 mutants was surprising to us, because previous work indeed observed that the cell fate defects in the DA neurons in the ventral nerve cord (DA2-DA7) in unc-4 mutants . One possible explanation is that the mechanisms of cell fate specification of these two posterior DA neurons are slightly different from the rest of DA and VA neurons. Consistent with this idea, DA8 and DA9 exhibit distinct gene expression profile compared with anterior DA neurons . In the revised manuscript, we described this possibility in the discussion.The lack of major cell fate defects in the DA8 and DA9 neurons of The downstream pathways  identified as important for tiling strongly overlap with those previously identified as important for neuronal fate determination. While perhaps not absolutely critical, identification of the proposed additional Wnt-regulated TF that is important (with ceh-12) for tiling might distinguish specific mechanisms for regulation of tiling and be a strong addition the story.ceh-12 and these factor(s), identification of these genes would be challenging. We hope to identify them through single cell RNA sequencing of DA8/9 in unc-4 mutants, as well as genetic suppressor screening of the synaptic tiling defects in the unc-4; ceh-12 mutants in the future. We included these future experiments in the revised discussion.We agree that identifying the additional transcription factor(s) that function downstream of Wnt signaling would greatly enhance our understanding of transcriptional regulation of precise synapse pattern formation. However, given the genetic redundancy between Related, has tiling been examined in VA neurons? Might unc-4/unc-37 have roles in both neuronal fate determination and later roles in synaptic tiling in these neurons? I don't expect the authors to resolve these questions experimentally, but a more direct discussion might help to clarify.mizIs3 (and wyIs446) are highly variable in wildtype, which made the phenotypic examination of unc-4 and unc-37 difficult. We hope to be able to answer this important question with a better VA-synaptic tiling marker in the future. In the revised manuscript, we have included a discussion regarding the potential functions of unc-4 and unc-37 in VA synaptic tiling and AVA-VA synaptic specificity.Our synaptic tiling marker is supposed to label two posterior VA neurons, VA11 and VA12, with GFPnovo2::RAB-3 and mCherry::RAB-3. However, the synaptic tiling between VA11 and VA12 labeled with


CSSR, an algorithm for assigning secondary structures to RNA 3D structures with missing atoms, has been developed. The base-pair assignment accuracy is close to 90% for 3D structures in which only one atom per nucleotide can be empirically identified. Coarse-grained Secondary Structure of RNA (CSSR), an algorithm for the assignment of rSS for structures in which nucleobase atomic positions are incomplete, has been developed. Using CSSR, an rSS assignment accuracy of ∼90% is achieved even for RNA structures in which only one backbone atom per nucleotide is known. Thus, CSSR will be useful for the analysis of experimentally determined and computationally predicted RNA 3D structures alike. The source code of CSSR is available at https://github.com/pylelab/CSSR.RNA secondary-structure (rSS) assignment is one of the most routine forms of analysis of RNA 3D structures. However, traditional rSS assignment programs require full-atomic structures of the individual RNA nucleotides. This prevents their application to the modeling of RNA structures in which base atoms are missing. To address this issue,  Traditional rSS assignment programs such as i.e. there are regions of the structure that are not known with certainty, or there are regions (or atoms) that are completely missing. For example, among the experimentally determined RNA structures deposited in the PDB, approximately 5.6% of the RNA chains only contain P atoms. Meanwhile, while there are a few programs such as FARFAR , the eight heavy atoms on the sugar ring  and the glycosidic N atom of the nucleobase. The rSS assignment is achieved by computing the agreement of pseudo-bond lengths, pseudo-bond angles and dihedral angles formed by constituent atoms between an input structure and the standard length/angle/dihedral values from statistics of canonical base pairs in high-resolution RNA structures. The CSSR program can be used for the ultrafast calculation of base-pairing energy terms during RNA folding and refinement simulations  and wobble (G:U) pairs. For each nucleotide pair i and j that satisfies these criteria, the CSSR score is calculated to indicate the base-pairing potential:For a given input atomic RNA structure, A = {P, C5′, C4′, C3′, C2′, C1′, O5′, O4′, O3′, N} is the set of atom types considered; diha, disa and anga are inter-nucleotide dihedral angles, inter-nucleotide distances and inter-atomic angles, respectively, between nucleotides i and j for atom type a as illustrated in Fig. 1Supplementary Fig. S1). If a certain dihedral/distance/angle cannot be calculated due to missing atoms, the respective term for the atom type is ignored for this nucleotide pair. In most RNA structures a base pair rarely exists as a singleton; instead, it is more commonly observed within helices, where the base pair can stack with a neighboring pair (or two neighboring base pairs) formed by adjacent nucleotides. Therefore, in CSSR, distances between i and j, between i + 1 and j − 1, and between i − 1 and j + 1 are all considered for each atom type. Meanwhile, the geometry definition of Here, 2.2.CSSR scores to remove conflicting base pairs. To this end, all nucleotide pairs with CSSR scores ≥0.5 are listed in descending order of their scores. Here, the CSSR score cutoff of 0.5 is chosen as it provides a good balance between precision and recall for almost all atom types . Nucleotide pairs are then iteratively excluded from this list if one or both nucleotides overlap with any pairs that rank higher on the list. The remaining pairs in the list will be the final base pairs assigned by CSSR. This post-processing step does not use dynamic programming such as that implemented by the Zuker  relative to the ground-truth assignment. These levels of agreements are 13%, 21% and 138% higher than those achieved by pdb2ss, which is the only existing rSS assignment program for coarse-grained RNA structures. Similar conclusions can be reached based on the Matthews correlation coefficient (MCC) instead of F1-score , Fold (F1-score = 0.610) and CycleFold (F1-score = 0.408).As shown in Fig. 2e Table 1. To put CSSR achieves a high agreement (F1-score = 0.944) with ground-truth assignment, which is derived by DSSR  and is comparable to the agreements among full-atomic rSS assignment programs , CSSR or pdb2ss. This is because the interatomic distance in a canonical base pair is farthest for the P atom compared with all other atom types . Consequently, the distances, dihedrals and angles calculated using P atoms have the largest variations , which makes rSS assignment challenging. We tested whether rSS assignment for the P atom can be improved by combining CSSR and RNAstructure through weighted averaging of their assignment/prediction scores, as these two programs are based on completely different principles. As shown in Table 1CSSR and RNAstructure, respectively, while the F1-score for other atom types show little to no improvement. Moreover, the inclusion of RNAstructure significantly slows down CSSR: for example, CSSR itself only needs 0.05 s for Lactococcus group II intron  but needs 18 s to include RNAstructure. Therefore, in this work, we use CSSR without RNAstructure as the default rSS assignment, although CSSR + RNAstructure is offered as an optional feature in the CSSR standalone program.It is more challenging to use the P atom than any other atom for rSS assignment by either CSSR assigns both Watson–Crick base pairs (A:U and G:C) and wobble base pairs (G:U), the accuracies of Watson–Crick pair assignments are consistently higher than those for wobble pairs for all atomic types (Supplementary Table S3). This is probably due to the much smaller number of wobble base pairs available in experimental structures that can be used to train CSSR . Similarly, due to limited training structures, the current CSSR method cannot assign Hoogsteen/sugar edge base pairs, which are even rarer than wobble base pairs. As more and more experimental RNA structures are determined, it is likely that a future version of CSSR retrained on more structures could improve the assignment accuracies for these non-Watson–Crick base pairs.While 3.3.CSSR to assign rSS to computationally predicted structures, which is one of the important motivations for developing CSSR. To this end, we collected all 21 modeling targets from a recent community-wide RNA puzzle challenge , even when using predicted 3D structure models as input, CSSR still achieves very high rSS assignment agreement with the native rSS . This level of agreement between native rSS and the rSS assignment for predicted structure models is similar to that achieved by existing full-atomic rSS assignment programs . This suggests the usefulness of CSSR even for low-resolution 3D structure models.We further examined the ability of RNA and r.m.s.d.) of the input 3D structure model, with Pearson correlation coefficients (PCCs) of −0.016 and 0.111, respectively . This is largely because RNA models with low global 3D structure quality can still have a high degree of rSS agreement with the native structure. As a case study, we examined the glycine riboswitch from RNA puzzle problem 3. The structure model has a TM-scoreRNA of 0.336 and an r.m.s.d. of 18.3 Å relative to the experimental structure . The main reason for the dissimilarity between the experimental and computationally determined structures is that the placement of the first 24 and last 12 nucleotides  was incorrect in the computational model, although the remaining 48 nucleotides adopted the correct topology . Despite an inaccurate 3D structure model, the rSS was largely modeled correctly , with only three missing base pairs and one incorrectly included base pair in the 3D model. Since the top RNA puzzle algorithms . This is much higher than that achieved by pdb2ss (F1-score = 0.495) and sequence-based rSS prediction by RNAstructure (F1-score = 0.697). These data confirm the applicability of CSSR to low-resolution experimental data.We further tested 4.CSSR, a new rSS assignment algorithm for detecting base pairs in RNA 3D structures. To our knowledge, CSSR is the one of only two algorithms available for rSS assignment in RNA 3D structures with missing atoms, and the only algorithm with 90% rSS assignment accuracy. The high accuracy of CSSR and its robustness, regardless of the input structure quality, makes CSSR a useful tool for modeling the base pairing within both experimental and computationally determined RNA structures. Moreover, the base-pairing score of CSSR (1et al., 2017et al., 2012et al., 2009CSSR focuses on the assignment of canonical base pairs. A natural extension would be the assignment of non-canonical base pairs. Work along this line is in progress.We developed 10.1107/S2059798322001292/cb5131sup1.pdfEvaluation metrics for rSS assignment, Supplementary Tables and Supplementary Figures. DOI:


Alternative cleavage and polyadenylation (APA), an RNA processing event, occurs in over 70% of human protein-coding genes. APA results in mRNA transcripts with distinct 3′ ends. Most APA occurs within 3′ UTRs, which harbor regulatory elements that can impact mRNA stability, translation, and localization.APA can be profiled using a number of established computational tools that infer polyadenylation sites from standard, short-read RNA-seq datasets. Here, we benchmarked a number of such tools—TAPAS, QAPA, DaPars2, GETUTR, and APATrap— against 3′-Seq, a specialized RNA-seq protocol that enriches for reads at the 3′ ends of genes, and Iso-Seq, a Pacific Biosciences (PacBio) single-molecule full-length RNA-seq method in their ability to identify polyadenylation sites and quantify polyadenylation site usage. We demonstrate that 3′-Seq and Iso-Seq are able to identify and quantify the usage of polyadenylation sites more reliably than computational tools that take short-read RNA-seq as input. However, we find that running one such tool, QAPA, with a set of polyadenylation site annotations derived from small quantities of 3′-Seq or Iso-Seq can reliably quantify variation in APA across conditions, such asacross genotypes, as demonstrated by the successful mapping of alternative polyadenylation quantitative trait loci (apaQTL).We envisage that our analyses will shed light on the advantages of studying APA with more specialized sequencing protocols, such as 3′-Seq or Iso-Seq, and the limitations of studying APA with short-read RNA-seq. We provide a computational pipeline to aid in the identification of polyadenylation sites and quantification of polyadenylation site usages using Iso-Seq data as input.The online version contains supplementary material available at 10.1186/s13059-021-02502-z. Although the human genome only harbors about twenty thousand protein-coding genes, the human transcriptome encodes ten times that number, or two hundred thousand unique transcripts . Over thThe cleavage and polyadenylation reaction is controlled by sequence elements upstream and downstream of the cleavage and polyadenylation signal (PAS) site. These sequence elements are recognized by several proteins and protein complexes that are recruited upon transcription of the PAS site by RNA polymerase II. Coordinated recognition of the signal site, a hexameric A[A/U]UAAA sequence or variant thereof, ~20–30 nucleotides upstream of the PAS site , 10, andThe rise of next-generation, high-throughput RNA sequencing (RNA-seq)  has alloSome existing approaches that leverage short-read RNA-seq to study APA rely on estimating PAU based on transcript-level abundance , 22 Figa. For exWhile these methods have provided valuable insights into the landscape of APA across a myriad of biological contexts, there are a number of challenges associated with studying APA with short-read RNA-seq. Generally, the estimation of isoform abundance from short-read RNA-seq is statistically challenging because short-read protocols tend to sample small portions of transcripts, and alternative transcripts often have substantial overlap . SpecifiBecause of the biases and analytical challenges associated with short-read sequencing protocols and their variants, we took advantage of the Pacific Biosciences (PacBio) single-molecule isoform-sequencing (Iso-Seq)  to more To define a set of PAS sites to benchmark against, we compiled eight polyA-selected PacBio Iso-Seq lymphoblastoid cell line (LCL) samples. Specifically, we generated five libraries for Yoruba (YRI) LCLs GM18501, GM18504, GM19144, GM19239, and GM19153  and obtaAligned reads containing polyA tails were extracted from the alignment files after performing a series of filtering steps, including filtering based on the length and adenosine composition of polyA tails and filtering out internal priming or mispriming to minimize false-positive PAS site identification Fig. a. In brip < 2.2e−6, Additional file p < 2.2e−6). Finally, we considered the distribution of the filtered set of reads across all genes in the genome and restricted the specific comparative analyses to 2862 genes with a read coverage of ≥ 40 Iso-Seq reads with a polyA tail to obtain a final set of 4446 high confidence PAS sites with PAUs > 5% . We also observed greater conservation upstream of PAS sites localized in 3′ UTRs than those in introns or other genic locations, which is consistent with previous findings , highlighting the importance of local chromatin architecture in PAS site selection , 78.7% were also defined as 3′-Seq PAS sites . This was expected given that different sequence isoforms can contain a significant amount of sequence overlap, and short RNA-seq reads assigned may be assigned to the incorrect isoform. Therefore, short-read-based methods may overestimate proximal PAU. Indeed, TAPAS and DaPars2 identified a significant number of distal PAS sites with lower PAUs; 74.7% of distal PAS sites identified by TAPAS exhibited PAUs ≤ 50%, and 61.6% of distal PAS sites identified by DaPars2 exhibited PAUs ≤ 50% , the furthest upstream  and furthest downstream PAS site . Interestingly, we observed little difference in proximal and distal PAUs identified by Iso-Seq and 3′-Seq . In contrast, TAPAS, DaPars2, QAPA, GETUTR, and APATrap exhibited significant PAU differences between proximal and distal PAS sites  curve to highlight the tradeoff between sensitivity and specificity of 3′-Seq, TAPAS, DaPars2, QAPA, GETUTR, and APATrap as compared to Iso-Seq PAS sites, which, for the purpose of this analysis, were considered to be the ground truth Fig. c. We didH3F3B as an example in which Iso-Seq and 3′-Seq identified five PAS sites (PAUs > 1%) with comparable PAUs , which link variations in PAU to genotype, using PAS sites defined by TAPAS, DaPars2, QAPA, GETUTR, and APATrap by benchmarking against apaQTL we identified using 3′-Seq data. We next ascertained whether the reliability and reproducibility of apaQTL called using a RNA-seq-based tool, QAPA, could be improved when fed custom PAS site annotations based on small quantities of specialized datasets, such as 3′-Seq or Iso-Seq.In order to map apaQTL, we restricted our analysis to samples with genotype information: 87 YRI LCLs for which we had access to short-read RNA-seq data and 51 YRI LCLs for which we had access to 3′-Seq data the “” sectionπ1 statistic and restricted to PAS-SNP pairs within 3′ UTRs given that some of the short-read RNA-seq-based methods do not identify PAS sites upstream of 3′ UTRs. Interestingly, very few apaQTL called using PAS sites defined by TAPAS, GETUTR, and APATrap were shared with apaQTL called by other methods , suggesting that running QAPA with Iso-Seq or 3′-Seq PAS site annotations derived from a small number of individuals is a reasonable alternative to exclusively performing 3′-Seq in many individuals for apaQTL mapping.To assess what fraction of apaQTL called by the various tools could be recapitulated by other tools, we estimated sharing of apaQTLs using Storey’s ods Fig. b, suggesods Fig. b. For exles Fig. . While tles Fig. . Interesπ1 between 0.09 and 0.36). In particular, sharing between eQTL and apaQTL identified using 3′-Seq (π1 = 0.36) is very similar to that between eQTL and apaQTL identified using QAPA run with 3′-Seq (π1 = 0.39) or run with Iso-Seq annotations (π1 = 0.36) , which can serve as a proxy for function. For example, shared apaQTL and eQTL include cases in which one PAS may provide more stabilility to a transcript over another, preventing the transcript from being subject to degradation. In such an example, high gene expression might serve as a proxy for transcript stability. Overall, we observed that sharing of apaQTL with associated gene-SNP pairs was indeed relatively high  across 12,280 genes, 38.7% of which are novel, using Iso-Seq data derived from eight LCL samples. We observed that APA detection methods, such as those that take short-read RNA-seq as input, including as TAPAS, DaPars2, QAPA, GETUTR, and APATrap, as well as 3’-Seq, were able to identify comparable numbers of PAS sites. Importantly, PAS sites identified by all methods exhibited well-characterized features of PAS sites, including enrichment of signal site motifs upstream of cleavage sites, enrichment within 3′ UTRs, and association with transcription elongation.We benchmarked the ability to study APA using with these methods against 3′-Seq and Iso-Seq. We estimated that 78.6% of PAS sites identified by 3′-Seq overlap with Iso-Seq-defined PAS sites whereas, at best, 56.6% PAS sites identified by one of the RNA-seq-based methods overlap with Iso-Seq-defined PAS sites. Moreover, as expected, there is reasonable concordance in the identification of PAS sites and estimation of PAUs between 3′-Seq and Iso-Seq. This is in contrast to the greater discordance between RNA-seq-based methods and Iso-Seq, likely because PAS site identification and PAU quantification among the RNA-seq-based methods can be highly variable. Overall, this suggests that researchers should carefully assess which RNA-seq-based method might serve them best based on the exact biological questions they may be interested in answering. Moreover, 3′-Seq should be the method of choice for studying APA when such data can be generated or are available.We acknowledge that it is not necessarily practical or cost-effective to generate specialized datasets to study APA, especially given that a plethora of short-read RNA-seq data are already publicly available. Through our analysis of inter-individual variation in APA as a test-case, it is apparent that QAPA, an isoform-based RNA-seq method to study APA, paired with PAS site annotations derived using small quantities of specialized sequencing data, such as 3′-Seq or Iso-Seq, may offer a considerable advantage in studying APA in a cost-effective manner in the near term, until it becomes more accessible and inexpensive to study APA extensively using full-length, long-read sequencing.This study demonstrates that current methods to study RNA processing events, such as APA, with short-read RNA-seq data suffer from limitations. However, combining large quantities fo RNA-seq data with small quantities of specialized data, in this case, 3′-Seq or Iso-Seq, strikes  an attractive balance between affordability and accuracy in the study of APA.2. These LCLs—GM18501, GM18504, GM19144, GM19239, and GM19153—were derived from the Yoruba (YRI) individuals from the International HapMap Consortium. The Coriell Cat #:Research Resource Identifiers (RRIDs) are GM18501:CVCL_P458, GM18504:CVCL_P460, GM19144:CVCL_P525, GM19239:CVCL_9634, and GM19153:CVCL_P531. These lines were authenticated and tested for mycoplasma contamination. Cell culture and RNA extraction were performed as described previously [5 cells, and grown to a concentration of 1×106 cells per mL before RNA extraction, which was performed as described previously [We cultured 5 Epstein-Barr Virus transformed lymphoblastoid cell lines (LCLs) at 37°C and 5% COWe processed a total of 8 polyA-selected PacBio Iso-Seq LCL libraries  Five SMRminimap2 -ax splice -uf -C5 hg19.fa <file>.fastq > <file>.sam. In order to increase power to call PAS sites, aligned reads from the eight libraries were pooled together.Reads were mapped to the hg19 human reference genome using minimap2 (version 2.2.15) separately for every library , using tTo identify Iso-Seq reads that capture cleavage and polyadenylation events, we searched for reads that contained stretches of adenosines . PolyA stretches needed to be located immediately after the 3′ end of the alignment . We assessed if the softclipped portion of every read contained a stretch of adenosines. We retained the reads if their softclipped segments were < 20 nucleotides in length and were composed of 95% adenosines. Moreover, if the length of the softclipped segment of a read was ≥ 20 nucleotides, we assessed if the first 20 nucleotides of the softclipped segment was composed of 80% adenosines and if the following 20 nucleotides of the softclipped segment was composed of 95% adenosines and retained these reads as containing a stretch of adenosines.Next, reads with stretches of adenosines were filtered for internal priming or mispriming using an approach similar to what has been described previously . In brieTo ensure the validity of our filtering steps, we verified that the set of final reads showed enrichment of hexameric polyadenylation signals , as described previously , 39, 40.Putative PAS sites were defined as the ends of the mapped portion  of reads and 100 nucleotides upstream. We then refined the set of reads used to define every putative PAS site by filtering out reads that did not map to annotated 3′ UTRs and that did not also span an upstream exon. We then refined this set of PAS sites by restricting to those in annotated genes using the annotatePeaks.pl script (HOMER v4.11) . This sc. For downstream analyses, we restricted to PAU > 5%  . Reads whttps://zenodo.org/record/1222196/files/qapa_3utrs.gencode.hg19.tar.gz), which is derived from GENCODE [Standard, short-read RNA-seq data for 89 LCLs  were obtained from the GEUVADIS project  , TAPAS [ GENCODE  and the Moreover, all RNA-seq based tools output estimates of PAU separately for every individual. Therefore, we averaged PAU across all individuals for downstream comparisons. The PAS sites were re-annotated with HOMER as described previously, and PAUs for every gene were re-scaled to sum to 1.0 if any PAS sites were omitted because they could not be annotated by HOMER. For downstream analyses, we restricted to PAU > 5%   to assesIn the analysis of sequence conservation, we used phyloP scores generated on the 46-way vertebrate alignment, restricting to placental mammals. These were downloaded from the UCSC Genome Browser .To assess the concordance between PAS site location and PAU quantification defined by the RNA-seq based tools and 3′-Seq as compared to those defined by Iso-Seq, we restricted to PAS sites that fell within the set of 2862 genes with Iso-Seq read coverage ≥ 40. In addition, we restricted to Iso-Seq PAS sites with PAUs > 5%.We directly assessed the overlap of PAS sites called by different methods using BEDTools . We defiROC curves were generated to assess the sensitivity and specificity of the RNA-seq tools and 3′-Seq in accurately identifying PAS sites. Specifically, the Iso-Seq PAS sites with PAUs > 5% were used as the ground truth. True positives are instances in which Iso-Seq PAS sites with PAUs > 5% have analogous PAS sites defined by other methods with PAUs > 0%. False positives are PAS sites defined by other methods with PAUs > 5%, but lack analogous PAS sites defined by Iso-Seq.We mapped apaQTL separately for all methods except Iso-Seq, for which we were lacking power to call QTL given our small sample size of eight individuals. For the RNA-seq methods, we removed two individuals, NA18500 and NA18908, due to low confidence in their annotated identity . For the 3′-Seq, we removed these same two individuals as well as NA19092 due to lack of genotype information . We analyzed all PAS sites defined by each of these methods, regardless of PAU.P value from a standard linear regression was extracted from the FastQTL output for every SNP-PAS pair. In addition, the lead SNP-PAS association for every PAS site was obtained from the 1000 permutations performed by FastQTL [We standardized all PAU measurements across individuals and then quantile-normalized them to fit a standard normal distribution, as described previously , 60. We  FastQTL . apaQTL , now with the molecular phenotype as gene expression instead of PAU. The same set of RNA-seq data from 87 individuals was used. The same set of SNPs, with MAF ≥ 0.05 were used, now within ± 1MB of genes.We mapped eQTL in a fashion analogous to apaQTLP value of the lead SNP-PAS pair from method A in method B [P value of the association between the lead SNP-PAS pair and gene expression level.To estimate sharing between apaQTL mapped using PAS site derived from different methods, we used Storey’s π1 method , which considers the method B . SimilarAdditional file 1: Fig. S1. Iso-Seq data filtering criteria for the study of APA. Fig. S2. Differential expression of alternative 3’UTRs between tissues. Fig. S3. PAS site genic, genomic, and usage features. Fig. S4. PAS site identification and PAU quantification across Iso-seq and short-read methods, including QAPA run with different PAS site annotations. Fig. S5. Comparison of apaQTL between APA methods. Fig. S6. Example of an apaQTL in the CASC3 gene defined by 3’-Seq exclusively.Additional file 2: Iso-Seq.PAS.sites. Iso-Seq PAS sites with PAU > 5% (BED format).Additional file 3: 3-Seq.PAS.sites. 3’-Seq PAS sites with PAU > 5% (BED format).Additional file 4: TAPAS.PAS.sites. TAPAS PAS sites with PAU > 5% (BED format).Additional file 5: DaPars2.PAS.sites. DaPars2 PAS sites with PAU > 5% (BED format).Additional file 6: QAPA.GENCODE.PolyASite.PAS.sites. QAPA (run with GENCODE and PolyASite annotations) PAS sites with PAU > 5% (BED format).Additional file 7: QAPA.Iso-Seq.PAS.sites. QAPA (run with Iso-Seq annotations) PAS sites with PAU ≥ 5% (BED format).Additional file 8: QAPA.3-Seq.PAS.sites. QAPA (run with 3’-Seq annotations) PAS sites with PAU ≥ 5% (BED format).Additional file 9: GETUTR.PAS.sites. GETUTR PAS sites with PAU > 5% (BED format).Additional file 10: APATrap.PAS.sites. APATrap PAS sites with PAU > 5% (BED format).Additional file 11: Review history.


Mononegavirales), was found to be a negative strand RNA virus in the 1960s, and since then the number of such viruses has continually increased with no end in sight. Sendai virus (SeV) was noted soon afterwards due to an outbreak of newborn pneumonitis in Japan whose putative agent was passed in mice, and nowadays this mouse virus is mainly the bane of animal houses and immunologists. However, SeV was important in the study of this class of viruses because, like flu, it grows to high titers in embryonated chicken eggs, facilitating the biochemical characterization of its infection and that of its nucleocapsid, which is very close to that of measles virus (MeV). This review and opinion piece follow SeV as more is known about how various mononegaviruses express their genetic information and carry out their RNA synthesis, and proposes a unified model based on what all MNV have in common.Vesicular stomatitis virus (VSV), the founding member of the mononegavirus order ( Rhabdoviridae (rabies virus and vesicular stomatitis virus (VSV)), Paramyxoviridae (SeV and MeV), Pneumoviridae  and human metapneumovirus (hMPV)) and Filoviridae (Ebola and Marburg viruses). Their non-segmented negative-strand RNA genomes are 11 to 19 kb in length with 5 to 10 tandemly arranged genes containing gene start (GS) and gene end (GE) signals that direct the synthesis of individual capped and polyadenylated mRNAs. Some genes can express more than one protein due to the presence of overlapping open reading frames via alternate ribosomal start codons and, perhaps uniquely for MNV, via co-transcriptional mRNA editing. This central protein-coding region is flanked by the replication promoters (or parts thereof) at the genome’s very 3′- and 5′-ends, termed 3′-leader and 5′-trailer, the latter being the complement of the 3′ end of the antigenome (The mononegavirus (MNV) order contains several families, including the tigenome .MNV genomes are never found as free RNA, but always enclosed within a non-covalent homopolymer of the viral nucleoprotein (N). This assembly of ssRNA and multiple copies of N, the nucleocapsid (NC), can be considered as the true MNV genome as only genome RNA within this structure is normally recognized by the viral polymerase (pol). MNV N cores are composed of two lobes and a positively charged groove in between where the RNA is bound. Each N subunit also has amino- and carboxy-terminal arms that dock with binding sites on adjacent protomers , formingIn contrast to positive-strand RNA viruses, negative-strand RNA viruses do not have nor need helicases, as their genomic RNA is always bound to N that binds only to ssRNA. However, they do need to avoid the possible annealing of genomic and viral mRNAs, especially newly formed ones. MNV NC assembly thus presumably starts at or near the genomic 5′ ends as they emerge from pol during replication, and for MeV, its N binds more tightly to the nt sequence at its 5′ end than that at its 3′ end . There iOD) (N/C-ter) that, except possibly for their extremities, are intrinsically disordered, providing great flexibility for their multiple interaction domains [MNV genome NCs are templates for two forms of RNA synthesis, that of mRNAs (termed transcription) and that of genome replication via antigenome NCs. MNV polymerases are complexes of the large L protein, responsible for all its catalytic activities, and a P (phosphoprotein) co-factor that oligomerizes due to a centrally located domain (POD) . The SeV domains ,16,17,18C-ter with N of the assembled NC (NNC) [core to twist relative to each other as pol moves down the NC during RNA synthesis, converting these displaced NNC from a closed to an open conformation in which RNA is no longer bound. This separation of N subunits from the template RNA and their replacement after the template has exited pol is presumably the rate-limiting step in MNV RNA synthesis.Although MNV nucleocapsids never disassemble once formed (as far as we know), their N protomers must be transiently separated from the genome RNA for the latter to enter pol’s template channel that leads past the synthesis chamber in pol’s core . The transient displacement of the Ns during MeV RNA synthesis is thought to be due to the transient and multi-faceted association of PNC (NNC) ,20,21. TRhabdo- and pneumoviruses have monopartite 3′ end promoters, i.e., all critical sequence elements for the initiation of RNA synthesis are within one segment of the leader region, mostly towards its 3′ end. In contrast, paramyxo- and filoviruses have bipartite promoters; the 3′-most promoter element (PE1) within leader, and PE2 within the 5′ UTR of the invariably first N gene . PE1 andAll aspects of paramyxovirus RNA synthesis are governed by the “rule of six”. All these genomes found in nature are a multiple of 6 nt long, and only minigenomes of hexamer length replicate well in cell culture. This rule imposes a hexamer phase on the entire genome, which is composed of a series of hexa-nt bound to each N subunit. Paramyxovirus PE2 simply consists of three contiguous hexamers where only one or two of these nt are at all important, and for SeV and PIV5 these nt are found in hexamer positions whose nt bases point towards the solvent . BecauseThe manner in which this bipartite promoter operates was examined for PIV2, a close relative of PIV5 and mumps virus. Ten residues of their N RNA-binding groove contact the RNA, nine with the ribose-PO4 backbone, and gln202 contacts a nt base . The RNAFiloviruses share many properties with paramyxoviruses, e.g., Ebola virus (EBOV) also employs mRNA editing, in this case to express its full-length receptor-binding protein, although there are also important differences. Bipartite promoters appear to be associated with MNV families that co-transcriptionally edit their RNAs . RecentlBoth plus-strand leader and minus-strand trailer RNAs  are founMechanistically, it is of course easier to separate the N chain from the genome RNA starting at a free 3’ end, than from TSS where both sides of the RNA chain are bound to nucleoprotein. For viruses with monopartite promoters such as VSV, their minigenome replication does not follow the rule of any integer. Here, dimethyl sulfate modification of nt bases within NCs, an indication of their accessibility to the solvent, occurs more readily and is independent of any integer phase, in contrast to that of SeV ,41,42. VSome MNV gene junctions are highly conserved, e.g., VSV, and SeV, whose intergenic regions (not copied into complementary RNA) are conserved and only 2–3 nt long. In contrast, the intergenic regions of other viruses are quite variable in length and sequence, even within different genera of the same family. In the latter cases, after terminating the upstream mRNA, transcriptases presumably scan the RNA within the NC and reinitiate mRNA synthesis upon encountering the next GS. Even for VSV and SeV, the 2–3 nt intergenic regions can be expanded by at least 200 nt without abrogating the ability of pol to specifically reinitiate mRNA synthesis when the ectopic GS is encountered. These studies have more accurately defined these GS, to be composed of at least the tail-end of the upstream GE sequence, the 2–3 nt intergenic region, as well as ca. the first 10 nt of gene start ,44, all High resolution structures of the VSV , PIV5 4, HMPV 4, PIV3 4 and RSV NC need to be transiently separated from the template RNA; the RNA-free N chain presumably traveling around L’s exterior and rejoining the template RNA as it exits pol  during mRNA synthesis and the editing site (underlined) is at the +1 position, the nascent chain of the RNA hybrid can slip backward relative to the template strand by one position before GTP can be incorporated. GTP is then added opposite the upstream C, adding a single pseudo-templated G to the nascent mRNA and bringing the editing site back to the original +1 position. For SeV and bPIV3, this editing cycle occurs at a specific frequency  and this difference is determined in part by the nature of the two nt directly upstream of the editing site . Appareor bPIV3 .0) is present in the cell, such as L, also bound to P4, via their N-ter tails [0 in P4-N0 is prevented from spontaneously assembling on irrelevant RNAs , N0′s sdroplets ,60, whicStructures of MNV L have identified five conserved domains: RNA-dependent RNA polymerase (RdRp), poly-ribonucleotidyltransferase (PRNTase that caps the mRNA 5′ ppp-end), connecting domain (CD), methyltransferase (MTase that further modifies the cap), and the C-terminal domain (CTD) ,47,48,500 depending on their availability. It is unclear whether P4 and P4-N0 affect L structure differently, but they do condition whether L acts as a transcriptase or replicase. All vRNA synthesis starts at the genomic 3′ end with 5′ ppp-leader (or trailer) sequences. For SeV, pol that have read through the leader/N junction terminate soon afterwards, whereas those having initiated N mRNA synthesis go through to the end of the gene [0/L replicase cannot, therefore, respond to cis-acting signals, and RNA synthesis only terminates when pol runs off the end of the template. In this scenario, termination of leader RNA synthesis is central to L acting as transcriptase. Notably, once fixed as a transcriptase or replicase, these pols cannot interconvert until either process has been completed. Transcriptases changing to replicases in mid-stream, or vice versa, would be highly counterproductive.When new L (devoid of P) is available , it can associate with either P4 or P4-Nthe gene . In the OD, and all  form pan detail ,47,48,50C-ter to “walk” along the N subunits of the N chain, or using more than two PC-ter to cartwheel on the N subunits of the NC. When SeV P was predicted to function as a coiled-coil oligomer, it was proposed that P acted by cartwheeling across this NNC assembly as L synthesized complementary RNAs, simply because there are more than two PC-ter tails. However, for PC-ter to cartwheel on the NC, all of P would also rotate, presumably while remaining bound to L. The recent P4-L structures offer a possible solution to the dilemma of how POD can rotate yet remain bound to L. In addition, they suggest a way whereby genome synthesis and its assembly into NCs can be tightly coupled, as the delay in adding even two protomers to the naked replicate RNA as it exits pol would expose 12 or more nt to which viral mRNA could anneal.P itself has no catalytic activity, and is thought to play a structural role in helping pol along the NC during RNA synthesis. L can progressively move down the NC guided by P in several ways, including using two POD may be less rigidly anchored due to PC-ter interactions that separate NNC protomers from the RNA template. Moreover, by super-positioning the individual RdRp domains of the two RSV structures (PDB: 6PZK and 6UEN), minor shifts between the interface of the L:P complex were identified, and Cao et al. have suggested that this interface may adopt a larger degree of conformational rearrangements during RNA synthesis [OD to rotate, each quarter turn accompanying each 6 nt incorporated. In both cases, POD of P4-L could then act as a rotating spindle, allowing its PC-ter to cartwheel across the NC. The same principle is applicable for filovirus P trimers, where each 120° turn accompanying each 6 nt is incorporated, and for rhabdoviruses, each 180° turn accompanying each 9 nt is incorporated.Pneumovirus P4 is bound to L by its parallel tetrameric coiled-coil, with two strands anchored to L’s surface. During RNA synthesis, however, Pynthesis . The pne0/L acts as a replicase and that its nascent chain synthesis and assembly are coupled , during its POD rotation the N0 bound to its PN-ter are available for their transfer to the emerging nascent genome RNA  but nevertheless carries out a remarkable number of different operations during transcription and replication. Given our very limited info about the conformational transitions that accompany these processes, the above scheme is far from the only one imaginable, but may help in elucidating the experimental pathways ahead.