Mouse nuclear RNAi-defective 2 promotes splicing of weak 5′ splice sites

  1. Matyas Flemr
  2. Michaela Schwaiger
  3. Daniel Hess
  4. Vytautas Iesmantavicius
  5. Josip Ahel
  6. Alex Charles Tuck
  7. Fabio Mohn
  8. Marc Bühler

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Abstract

Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5′ splice site (5′SS). In mammals, many introns contain weak 5′SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5′SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5′SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5′SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing.

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  1. Version published to 10.1261/rna.079465.122
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    Referee #3

    Evidence, reproducibility and clarity

    In this study the authors present the discovery of two new splicing factors NRDE2 and CCDC174 that interact with the U1 snRNA and with 5' splice sites and modulate usage of 5' splice sites with generally weaker pairing potential to the U1 snRNA. They develop a new cross-linking technique BCLIP for monitoring RNAs interacting with a particular protein by deep sequencing, which modifies the classical CLIP protocol and appears to allow them to detect interactions with proteins of low abundance such as NRDE2. They propose that NRDE2 and CCDC174 may form alternative U1 snRNP complexes distinct from the canonical U1 snRNP, which may be partly responsible for selecting alternative, weaker 5'SS.

    The study provides a plethora of experiments that provide strong experimental support for a model in which NRDE2 interacts with the U1 snRNA, recruits CCDC174, and together they tend to promote correct usage of weak 5' splice sites that are often flanked by several weak, cryptic 5' splice sites. The RNA-Seq supports a genome-wide role for NRDE2 in promoting splicing of weaker 5'-splice sites, while the in vivo reporter assays are elegant experiments showing a role for NRDE2 in enforcing correct usage of the most upstream weak 5'splice site.

    While the authors provide strong evidence in support of the main model proposed in their discussion, there are a few significant matters that are not addressed. Firstly, the fact that only a small proportion of 5'SS bound by NRDE2 appears to be sensitive to NRDE2 KO, even when translation-linked surveillance is blocked by cycloheximide, raises the possibility that the RNA-Seq technique may miss a significant proportion of transcripts that are unspliced and are rapidly degraded, potentially co-transcriptionally, by the nuclear exosome in a manner that may not necessarily depend on MTREX. Given that a significant proportion of unspliced transcripts may follow such a pathway (reviewed in Gordon et al., Curr. Op. Gen. and Dev. 2021), the authors should at least consider this possibility in their presentation of results and discussion. Ideally one could try to combine rapid depletion of NRDE2 with depletion or partial inactivation of one of the nuclear exosome components RRP6 or RRP44, although this reviewer recognizes that this may be technically challenging and lead to indirect effects on cell growth that might confound the analysis. Sequencing of specifically nascent RNAs associated with Pol II from a chromatin fraction, might offer a way to uncover additional NRDE2-sensitive transcripts.

    Secondly, the fact that NRDE2D200 shows a massive increase in U1 snRNP reads by the BCLIP procedure potentially suggests that NRDE2 may actually be part of a surveillance pathway to enforce usage of specific 5'SS and minimise cryptic 5'SS use. In this model, NRDE2 might bind all 5'SS but needs to be dissociated from the U1 snRNP either before or during 5'SS transfer by a helicase (e.g. MTREX or DDX5) within a certain time frame to prevent transfer of cryptic 5'SS. This model would be reminiscent of the initial binding and subsequent dissociation of Mud2 by Sub2 during E complex formation in yeast or of U2AF by DEK during proofreading of initial 3'SS recognition in humans. The fact that targets sensitive to NRDE2, as judged by RNA-Seq and expression profiling, mostly do not overlap with those MTREX, does not exclude the possibility that NRDE2 may act in cooperation with MTREX to prevent usage of cryptic 5'SS, which might result in production of rapidly degraded unspliced transcripts that are not detectable by the RNA-Seq methodology used here. Minimally, this reviewer thinks this possibility needs to be considered and briefly discussed by the authors.

    Finally, although provocative, the idea that NRDE2 binds an alternative U1 snRNP is not necessarily implied by the data. The fact that U1A, U1C, and U1-70k are not detected by MS in a tandem IP set-up that uses disrupts RNA structure and potentially protein-RNA interactions cannot be considered clear evidence for an alternative snRNP. Structures of the U1 snRNP suggest that association of such auxiliary proteins may depend on the structure of the U1 snRNA. The authors need to either modulate and clarify their claim, or provide stronger evidence, e.g. from more gentle IPs with U1A and U1-70K that U1 snRNPs that associate with these factors are not also associated with NRDE2. Related to this, this reviewer thinks it is tenuous to claim that the harsher xTAP-MS analysis involving formaldehyde is more indicative of "native" interactions because it uncovers binding of one core spliceosomal component and of TFIP11 and DHX15.In fact, the opposite seems more likely, that such reported interactions are not indicative of direct proximity, but rather result from perturbations to the native RNP structure induced by benzonase. In this sense, the claim that these observations suggest an "alternative" spliceosome assembly pathway seems particularly problematic, especially in view of the fact that in vitro studies suggest that DHX15 can associate with the U2 snRNP and can disassemble complexes at all stages of spliceosome assembly and catalysis, including during the pre-catalytic stage. The authors should be more careful with their wording and interpretation here.

    Depending on how the authors address the issues raised above, and how they modify their claims in the text, additional experiments may be deemed beyond the scope of the present study and should not be strictly necessary for publication, with the likely exception of the issue of alternative U1 snRNPs, where additional IPs might clarify, and potentially strengthen, the authors' claims.

    Significance

    This reviewer is an expert in the biochemical and structural study of pre-mRNA splicing and considers the present study an important contribution to understanding 5' splice site usage in higher eukaryotes. While the observation that spliceosomes from higher eukaryotes use additional protein factors to modulate 5' splice site selection is not new, the discovery of specific factors bound to U1 snRNA that may directly affect its binding to stronger or weaker 5'SS is certainly novel and of potentially broader significance. Although the author's claim that this may reflect alternative U1 snRNPs is not fully supported by the evidence presented, the proposal itself is an important potential advance, if it holds up to more stringent testing. The potential for NRDE2 to be part of a more complex surveillance mechanism to enforce use of specific 5'SS, which the data may also point to, would be an equally important advance. Finally, the observed interactions with U4 and U6 snRNAs, on which the authors do not comment much, provide further support for the idea that transfer of the 5'SS from U1 to U6 may be a particularly crucial step in 5'SS selection that is modulated in higher eukaryotes by non-canonical factors. Indeed, this latter point is also a significant contribution of the present study.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Flemr and colleagues describe novel functions of the splicing-associated proteins NRDE2 and CCDC174. These two proteins have previously been implicated in splicing (Jiao et al., RNA, 2018) and interact with the helicase Mtr4 and the exon-junction complex (Richard et al., RNA, 2018), which targets RNAs for degradation by the exosome. Here, the authors use a combination of genomics, including a modified CLIP protocol, genetics, and mass spectrometry to establish two key findings: (1) NRDE2 and CCDC174 act in concert to promote pre-mRNA splicing from non-consensus 5' splice sites (5'SSs) and (2) together with U1 snRNA they form a novel non-canonical U1 snRNP. The data themselves are clearly presented and of very high quality, but I do not agree with the authors interpretations and the two key claims.

    On claim (1), instead of NRDE2/CCDC174 specifically and actively promoting the usage of correct weak 5'SSs, they could alternatively promote correct 5'SS choice indirectly, by suppressing nearby cryptic 5'SSs. This is consistent with known functions of the EJC (e.g. Boehm et al., Mol Cell, 2018), with which NRDE2 and CCDC174 interact (Jiao et al., RNA, 2018) and their known association with Mtr4, which is also re-produced in the manuscript. This is further supported by the authors data, which shows that NRDE2 and CCDC174 CLIP signal peaks at the same sites as EJCs, upstream of 5'SSs. Prior to publication the authors should experimentally distinguish between active (direct) and passive (indirect) 5'SS selection mechanisms by NRDE2 and CCDC174.

    On claim (2), a new U1 snRNP would be a major discovery, yet, given the presented data, this conclusion should either be removed completely from the manuscript or needs to be rigorously tested. See comments below.

    Major comments

    1. The authors show an enrichment by MS-IP of NRDE2 in Fig 1A, 1C (the improvied xTAP-MS protocol) for late-stage spliceosome components, such as TFIP11 that is required for spliceosome disassembly (ILS complex), consistent with earlier data in C. elegans (Jiao et al., RNA, 2018). Given the consistency of the late stage-spliceosome interaction and the EJC with published results, how do the authors reconcile the proposed functions in 5'SS selection with known interactions of NRDE2 and CCDC174 with the EJC and disassembling spliceosomes? If NRDE2 and CCDC174-U1 formed, they would dissociate from the spliceosome with U1 snRNA during the Prp28-dependent pre-mRNA handover from U1 to U6 snRNA. How would NRDE2 and CCDC174 re-associate after the subsequent Pre-B to B to Bact to C* transitions in C, when the EJC binds the spliceosome, or after the subsequent C to P to ILS transitions in the ILS, when e.g. TFIP11 binds. In a more likely model, early and late splicing factors co-IP in the authors MS experiments because splicing factors are enriched generally with eachother and in e.g. nuclear speckles. Perhaps more stringent washes in the xTAP-MS experiment could home in on more direct interactions of NRDE2 or CCDC174 to the spliceosome?
    2. The following comments relate to the claim of a non-canonical U1 snRNP.
      • Fig. 6B: to assess the predictive power of the CLIP signal to reveal protein-snRNA interactions, can the authors comment on the expected crosslink efficiency and specificity of a bona fide U1 snRNP protein to U1 or a U2/U4/U5/U6 snRNP protein to its respective snRNA as well as all other snRNAs? How would these efficiencies and specificities compare to NRDE2-U1?
      • Fig. 6C: Can the relative differences in snRNA abundance, U1 being the most abundant, explain the CLIP crosslink efficiencies without the requirement of a bona fide NRDE2-U1 complex?
      • In Fig. 6C, have the authors looked at other spliceosomal snRNAs and their enrichments in the northern?
      • Fig. 6G, did the authors measure cellular snRNA levels after SmE dTAG depletion? The prediction would be that all snRNAs are reduced in steady-state abundance, due to improper biogenesis, which could explain why the U1 snRNA CLIP-seq signal is reduced. This would be independent of an NRDE2-U1 interaction.
      • As it would be surprising and exciting, if U1A, U1C, and U1-70k were absent from a functional U1 snRNP, this requires additional proof. Can they authors use an anti-U1 snRNA oligo in tandem with the NRDE2 IP or CCDC174 IP to show that the Sm-ring proteins and U1 snRNA are highly enriched but not U1A, U1C, and U1-70k proteins or any other snRNA?
      • U1C provides a ZnF domain that stabilizes the pre-mRNA 5'SS in its binding to U1 snRNA (Kondo et al., Elife, 2015). U1-70k stabilizes the U1 snRNP (Kondo, Elife, 2015) and can couple to RNA polymerase II (Zhang et al., Science, 2021), and is important for U1 snRNP biogenesis (Byung Ran So et al., NSMB, 2016). How would NRDE2 or CCDC174 compensate for these essential activities? Given the various crucial functions known U1 proteins perform, the claim that NRDE2 or CCDC174 can substitute them, should be supported by proof of their functional substitution.
      • Since NRDE2 or CCDC174 and U1 snRNA would be conserved and presumably for a high-affinity complex, ideally the authors would provide biochemical proof of their interactions, though this is may be beyond the scope of the current manuscript.

    Minor comments:

    • The conditions of the dTAG experiment are insufficiently described, what was the efficiency of depletion (Western blots or mass spec?) and over which time-scale was this applied?
    • Introduction, in the sentence '[...] encode much shorter U1 snRNA [...]' the authors imply that longer U1 snRNAs are correlated with a lack of splice site degeneracy. Yet, structural and mechanistic data show that the expanded U1 snRNA segments in e.g. S. cerevisiae (or U2 snRNA, which contains a 1000 nt) insertion are distant from the U1 snRNA 5'-end that recognizes the 5'SS or the U2 snRNA BSL, which binds the branch site, and thus are unlikely to influence splice site selection. Please rephrase.

    Significance

    NRDE2 and CCDC174 are enigmatic proteins that are likely to function during mRNA biogenesis and both have been linked to splicing and RNA decay. It is thus interesting to understand their precise modes of action. While the authors provide excellent data, the conclusions are not substantiated. I have expertise in the mechanistic study of pre-mRNA splicing, based on which several of the authors claims such as a new U1 snNRP complex, are challenging to reconcile with the past decades of splicing research. Given the sizable impact a new U1 snRNP would have on the field, these data must be unimpeachable.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Flemr et al. characterize the roles that proteins Nrde2 and CCDC174 play in mammalian splicing regulation. The authors perform native (nTAP-MS) and cross-linking (xTAP-MS) based IP-MS methods to identify the NRDE2 and NRDE2 mutant interactomes, and demonstrate NRDE2's enriched interactions with splicing factors. The authors further develop a RNA footprinting method (BCLIPseq) in order to capture NRDE2 and CCDC174 binding patterns on RNA, revealing a preference for binding unspliced introns close to the 5' SS. Furthermore, upon NRDE2 knockdown, the authors note a significant increase in alternative 5'SS splicing events, making NRDE2 a putative regulator of cryptic 5'SS. Following up on this observation through luciferase reporter assays, the authors demonstrate how NRDE2 and CCDC174 work together to inhibit cryptic splicing at weak 5'SSs. Finally, the authors demonstrate that NRDE2 and CCDC174 interact with U1 snRNA (but not core U1 snRNP protein components), providing a basis for their interactions with 5'SS. Overall, the authors thoroughly characterize protein and RNA interactions with NRDE2, demonstrating its role in mammalian pre-mRNA splicing, and its concerted role with CCDC174 in regulating splicing at some weak 5' splice sites.

    The study would be greatly improved through additional controls and more careful analyses. For instance, many controls are missing for the cell lines used throughout the study, making interpretation of the data more difficult. Other issues include that techniques developed within the study are presented without validation experiments, and key analyses, including microscopy experiments and rMATs analysis of RNAseq data, are performed without proper quantification, weakening the authors' conclusions from these experiments. Finally, major conclusions of the paper, such as the potential role of NRDE2 in a non-canonical U1 snRNP complex, would be greatly strengthened by additional experiments. However, overall, it appears that many of the major concerns should be readily addressable.

    Major comments

    1. Data are not present to demonstrate that cell lines were validated and compared properly:
      • a. The authors say "We assessed potential consequences of the tagging approach on protein function by comparing RNA-seq gene expression profiles with that of untagged cells, which remained unchanged for the proteins we report hereinafter" (p.6). This analysis should be made available in the supplement. They should additionally show western blots of tagged/untagged protein, in order to demonstrate similar expression levels for endogenous and engineered proteins.
      • b. Knockdown (KD) levels of all proteins from engineered lines should be shown over the KD timeline used in the study. For instance, no westerns in the paper show the degradation efficiency of the dTAG KD lines.
      • c. The authors should address why knockdown lines were made in different ways for different proteins (ex. only one cell line is a dTAG degradation line) and why knockdowns were performed for different amounts of time for every protein.
      • d. The authors should consider that, since knockdowns are performed for different amounts of time, results between protein knockdowns may not be directly comparable. For instance, in figure 3D, Ccdc174 dTAG lines have less misspliced target introns than the other knockdown lines. However, this may simply be because the knockdown period is shorter for the dTAG line than the other knockdown lines, and the length of treatment affects the overall number of introns affected.
    2. Nuclear localization experiments would benefit from further controls and quantification:
      • a. The authors conclude that "NRDE2 localisation to nuclear speckles depends on active pre-mRNA splicing" (p.7), which seems to contradict their result that "Chemical inhibition of splicing with Thailanstatin A (Liu et al., 2013) resulted in...wild-type NRDE2 remaining concentrated in enlarged NSs (Figure 1H and S1J)" (p.8).
      • b. Since the splicing inhibitor Thailanstatin A also changes the localization patterns of U2AF2 (Fig. 1G-H), it is unclear if U2AF2 is still a reliable nuclear speckle marker in the presence of the drug. Additional controls (such as staining for other nuclear speckle markers) are necessary to make this assertion.
      • c. To make the conclusion that "NRDE2-D174R accumulated in nucleoli" (p. 8), the authors should also include a nucleoli marker in their microscopy experiments.
      • d. Signal quantification of NRDE2 distribution/overlap with U2AF2 signal would strengthen the conclusions in Fig. 1G-H.
      • e. Quantification would again be helpful in Fig. 5C to demonstrate changes in NS localization. In addition, it looks like Nrde2-KO does not just lead to lack of CCDC174 accumulation, but to a decrease in its overall expression. The authors should comment on this observation, or quantify CCDC174 signal in both images to demonstrate that the overall levels remain the same.
    3. Since BCLIPseq is a technique developed by the authors, a more in-depth discussion of the technique development and quality control of the resulting data is warranted.
      • a. The authors mention that BCLIPseq offers a "streamlined and sensitive alternative to existing CLIP techniques" (p.9), but they don't provide any specifics into the ways they improve existing CLIP techniques in the main text. In what ways is it more streamlined and sensitive? This should be discussed in the main text rather than just the discussion, in order for the assertions made to be backed up with (supplementary) figures. A comparison of the coverage provided by a BCLIPseq library for NRDE2 to a CLIP library, for instance, would help to support these assertions.
      • b. The authors should address or provide evidence for why on-bead polyadenylation is preferable/more efficient than adapter ligation, especially as polyadenylation may be variable across transcripts. For instance, the authors could show more controls demonstrating the efficiency of on-bead polyadenylation or cite papers that have already extensively tested on-bead polyadenylation.
      • c. Many other RNA footprinting techniques (eCLIP, RIPseq) have noted significant nonspecific background in the resulting libraries, and usually use input controls to filter for this nonspecific background. The authors should clearly state if their BCLIPseq libraries also suffer from the same nonspecific background, and if so, what quality control steps exist in their analysis pipeline to minimize this background.
      • d. Related to the previous point, there is a high amount of rRNA reads in all the BCLIP libraries except EIF4A3. The authors suggest it is likely background, but if they are using a FLAG antibody for all of these, I'm not sure why there would be so much more background for some and not the others. If it's because EIF4A3 pulls down much more RNA with it because it binds most exon-exon junctions, whereas binding of the others is more rare, then isn't it possible that the mRNA reads are also partially background? This could explain why there is a very small overlap between the BCLIP bound loci and the affected 5'SS. An input control would help to determine what is indeed background.
    4. The conclusion that "This [U1 snRNA binding] leads to the provocative idea that NRDE2 could potentially mediate the formation of a non-canonical U1 snRNP" (p.20) is a very intriguing conclusion that would largely benefit from additional experiments to strengthen the claim.
      • a. Depletion of a U1-specific protein (U1-70k, U1C) and analysis of the effect (or lack thereof) on Nrde-U1 snRNA interactions would strengthen the assertion that Nrde-U1 snRNA interactions are independent of core U1 snRNP components.
      • b. Depletion of a U1-specific protein (U1-70k, U1C) and analysis of the effect (or lack therefore) on Nrde2-KO sensitive introns would also strengthen the assertion that Nrde2 regulates introns as part of a non-canonical U1 snRNP.
      • c. Overall, a schematic in the last figure that depicts the splicing model presented in the discussion would be helpful for describing the Nrde2/Ccdc-174 model proposed.
      • d. The authors show that the majority of ms-snRNA reads map to U1 snRNA. However, U1 snRNA is generally more abundant than other snRNAs (Dvinge et al. 2019), so the authors should show how the distribution shown in Fig. 6B compares to the input distribution of snRNA levels in the cell line used. Also, relative levels of U1 snRNA detected by IP-Northern Blot (Fig. 6C) don't seem to match the results shown in Fig. 6A and B, as U1 snRNA seems most abundant in the NRDE2 IP by Northern Blot and most abundant in NRDE-Δ200 by BCLIP-seq.
      • e. In Figs. 6D, E and F, the authors suggest that NRDE2 and CCDC174 contact U1 snRNA at multiple positions based on observing highest enrichment over SL2 and SL3. However, snRNAs are highly structured and modified, which may interfere with reverse transcription during library preparation and lead to uneven signal throughout the gene body. To show that the proteins of interest are really enriched at these positions, the authors could perform the same experiment for a protein that is known to bind at a different location on U1 snRNA.
    5. The rMATs analysis performed is very lenient; notably, there is no reported filtering for splicing events with some minimum coverage across replicates, and the inclusion level difference threshold of >0 (rather than >0.1 etc) is extremely low. As the rMATs analysis is key to the authors conclusion that there is "frequent cryptic 5'SS upon Nrde2 knockout" (p. 13), it seems important that this analysis is performed with more stringency in order to capture robust and meaningful splicing changes.

    Minor comments

    1. Some parts of the paper are organized in a confusing manner:
      • a. It is unclear why development of the xTAP-MS protocol is under the section "NRDE2 Localization to Nuclear Speckles Depends on Active pre-mRNA Splicing"
      • b. The section "Nrde2 and Mtrex Knockouts Induce a 2C-like State", while interesting, seems to be outside the scope of the paper
    2. It would be interesting for the authors to look into the BCLIPseq data to see if there are any enriched RBP binding motifs for the proteins studied.
    3. Western blots for IPs (ex. Fig 1F) should show the input for both the IP bait and prey proteins, not just the prey. In addition, input and IP'ed protein should be displayed in the same western blot image (without cropping in-between).
    4. Previous studies (Boehm et al 2018) have found that other EJC-associated proteins also are important for regulation of 5' cryptic splice site usage. It would be interesting for the authors to compare the 5' cryptic splice sites identified in these earlier studies to look for overlap between the 5' cryptic splice sites regulated by these proteins vs. NRDE2.
    5. The luciferase reporter assays are an especially strong portion of the paper, and are a nice orthogonal validation of the link between Nrde2, splicing regulation, and SS strength.
    6. It would be interesting for the authors to investigate the effect of the mutations in the luciferase reporter constructs on the binding patterns of Nrde2 on construct transcripts. This may help provide a mechanistic basis for the chosen cryptic 5' SSs.
    7. "Thus, NRDE2 promotes splicing from the most upstream of a series of 5'SSs" (p. 16) is an interesting conclusion, but this statement is far too general given the low number of genes surveyed using the luciferase assay. The statement should be rewritten to reflect that this statement has only been shown to be true for the few genes tested.
    8. The statement "NRDE2 and CCDC174 promote splicing at many of the same weak 5'SSs" (p. 16) would be stronger if it was not just based on the genes studied through the luciferase assay, but based on splicing changes analyzed genome-wide through rMATs analysis. Do NRDE2 and CCDC174 promote splicing of the same weak splice sites globally?
    9. R squared values should be added to the correlations presented in Fig. S2B to support the claim that replicates "correlated strongly", since that is the basis for merging replicates in subsequent analyses.
    10. In Fig. 3A, the four clusters should be annotated on the figure to increase clarity.

    Significance

    Overall, the study is thorough in its approaches to studying NRDE2 biology and makes a strong case for the exciting role of NRDE2 and CCDC174 in 5'SS selection. Combined IP-MS and RNA footprinting approaches compellingly demonstrate NRDE2's associations with splicing factors and splice sites in vivo. In addition, the combination of genome-wide approaches (RNAseq) with targeted analyses (luciferase reporter assays) allow for detailed analyses of cryptic splice site choice in the absence of NRDE2, NRDE2 mutants, MTREX, or CCDC174. These experiments support the novel role of NRDE2 and its associated proteins in splice site choice.

  6. Version published to 10.1101/2022.01.25.477700v1 on bioRxiv