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Model of the complete human U1 snRNP.a, Overview of a model of the complete U1 snRNP. Truncated SL2 was extended with A-form RNA and the crystal structure of the U1A–RNA complex19 was appended to the extended helix. The internal loop of SL2, consisting of four consecutive non-Watson–Crick base pairs (red), is in a position to interact with Sm-B and Sm-D1. b, Two views of the complete U1 snRNP model approximately 45° apart, with surface representation superimposed. Closely matching images are found in the gallery of negatively stained images of U1 snRNP reported previously23, 24.
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Human spliceosomal U1 small nuclear ribonucleoprotein particles (snRNPs), which consist of U1 small nuclear RNA and ten proteins, recognize the 5' splice site within precursor messenger RNAs and initiate the assembly of the spliceosome for intron excision. An electron density map of the functional core of U1 snRNP at 5.5 A resolution has enabled us...
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Context 1
... consists of two RBDs linked by a proline-rich sequence, the N- terminal RBD binds to the ten-nucleotide loop of SL218,19. In order to complete the model of U1 snRNP, the crystal structure of the U1A-RNA complex19 was added onto an extended SL2 helix (Fig 5a). Interestingly, the internal loop in SL2 consisting of four conserved non- canonical base-pairs (Fig 1S) would be in a position to interact with B and D1 on the rim of the Sm ring (Fig 5a). ...
Context 2
... order to complete the model of U1 snRNP, the crystal structure of the U1A-RNA complex19 was added onto an extended SL2 helix (Fig 5a). Interestingly, the internal loop in SL2 consisting of four conserved non- canonical base-pairs (Fig 1S) would be in a position to interact with B and D1 on the rim of the Sm ring (Fig 5a). This interaction could further stabilize the RNA structure onto the core domain. ...
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Genomic variation in the untranslated region (UTR) has been shown to influence HLA class I expression level and associate with disease outcomes. Sequencing of the 3'UTR of common HLA-A alleles indicated the presence of two polyadenylation signals (PAS). The proximal PAS is conserved, whereas the distal PAS is disrupted within certain alleles by seq...
Citations
... Eventually, snRNPs assemble at pre-mRNA splice sites and facilitate the removal of introns. In most cases, soon after it is transcribed, the pre-mRNA 5'-splice site (5'ss) at the beginning of an intron is recognized by the U1 snRNP and the intron branchpoint sequence within the intron is then recognized by the branchpoint binding protein and the U2 snRNP [21,[26][27][28]. Following the recruitment of the U4/U6.U5 tri-snRNP and a series of rearrangements whereby the U1 and U4 snRNPs are removed from the splicing complex, the branchpoint, 5'ss, and 3'ss are brought into closer proximity and a splicingcompetent (i.e. ...
... is an evolutionarily conserved RBP that has previously been implicated in poly(A) tail length control, transcriptional elongation, and mRNA splicing [26,[59][60][61][62][63][64][65]. Mutations in ZC3H14 have been linked to human intellectual disability [60], liver cancer [66], and tauopathy [67,68]. ...
... ZC3H14 and the associated THO components THOC1 and THOC5 coordinate the expression of several shared mRNA targets within neurons, including PSD95, a component of the post-synaptic density [65]. ZC3H14 is localized within nuclear speckles, the proposed storage sites for multiple spliceosome components [24,26], and loss of ZC3H14 also causes the accumulation of unspliced pre-mRNA in the nucleus [62]. The yeast and fruit fly orthologs of ZC3H14, called Nab2 (and in some studies, dNab2), have also been implicated in both poly(A) tail length control and pre-mRNA splicing [61,63,69,70]. ...
The regulation of cell-specific gene expression patterns during development requires the coordinated actions of hundreds of proteins, including transcription factors, processing enzymes, and many RNA binding proteins (RBPs). RBPs often become associated with a nascent transcript immediately after its production and are uniquely positioned to coordinate concurrent processing and quality control steps. Since RNA binding proteins can regulate multiple post-transcriptional processing steps for many mRNA transcripts, mutations within RBP-encoding genes often lead to pleiotropic effects that alter the physiology of multiple cell types. Thus, identifying the mRNA processing steps where an RBP functions and the effects of RBP loss on gene expression patterns can provide a better understanding of both tissue physiology and mechanisms of disease.
In the current study, we have investigated the coordination of mRNA splicing and polyadenylation facilitated by the Drosophila RNA binding protein Nab2, an evolutionary conserved ortholog of human ZC3H14. ZC3H14 loss in human patients has previously been linked to alterations in nervous system function and disease. Both fly Nab2 and vertebrate ZC3H14 bind to polyadenosine RNA and have been implicated in the control of poly(A) tail length. Interestingly, we show that fly Nab2 functionally interacts with components of the spliceosome, suggesting that this family of RNA biding proteins may also regulate alternative splicing of mRNA transcripts. Using RNA-sequencing approaches, we show that Nab2 loss causes widespread changes in alternative splicing and intron retention. These changes in splicing cause alterations in the abundance of protein isoforms encoded by the affected transcripts and may contribute to phenotypes, such as decreases in viability and alterations in brain morphology, observed in Nab2 null flies. Overall, these studies highlight the importance of RNA binding proteins in the coordination of post-transcriptional gene expression regulation and potentially identify a class of proteins that can coordinate multiple processing events for specific mRNA transcripts.
Author Summary
Although most cells in a multicellular organism contain the same genetic material, each cell type produces a set of RNA molecules and proteins that allows it to perform specific functions. Protein production requires that a copy of the genetic information encoded in a cell’s DNA first be copied into RNA. Then the RNA is often processed to remove extra sequences and the finalized RNA can be used to create a particular type of protein. Our work is focused on how cells within developing fruit fly brain control the types, processing steps, and final sequences of the RNA molecules produced. We present data showing that when fly brain tissue lacks a protein called Nab2, some RNA molecules are not produced correctly. Nab2 loss causes extra sequences to be retained within many RNA molecules when those sequences are normally removed. These extra sequences can alter protein production from the affected RNAs and appear to contribute to the brain development problems observed in flies lacking Nab2. Since Nab2 performs very similar functions to a human protein called ZC3H14, these findings could provide a better understanding of how ZC3H14 loss leads to human disease.
... Within this intricate process, nascent pre-mRNA undergoes 5'capping, splicing, and 3'-end processing, each step meticulously orchestrated to contribute to cellular homeostasis and fitness maintenance. [1] Comprising the U1 snRNA, a heptameric ring of Sm proteins, and three U1-specific proteins (U1-A, U1-C, and U1-70K), the three-dimensional organization of U1 snRNP, as revealed by its crystal structure, [2] underscores its functional prowess in mRNA splicing. U1 snRNP serves as a seeding particle during spliceosome assembly, binding to and defining the 5'-splice site by directly hybridizing the 5'-end of the U1 snRNA with the site, [3] a pivotal and limiting step in the splicing reaction, critical for splicing regulation. ...
The U1 small ribonucleoprotein (U1 snRNP) plays a pivotal role in the intricate process of gene expression, specifically within nuclear RNA processing. By initiating the splicing reaction and modulating 3’‐end processing, U1 snRNP exerts precise control over RNA metabolism and gene expression. This ribonucleoparticle is abundantly present, and its complex biogenesis necessitates shuttling between the nuclear and cytoplasmic compartments. Over the past three decades, extensive research has illuminated the crucial connection between disrupted U snRNP biogenesis and several prominent human diseases, notably various neurodegenerative conditions. The perturbation of U1 snRNP homeostasis has been firmly established in diseases such as Spinal Muscular Atrophy, Pontocerebellar hypoplasia, and FUS‐mediated Amyotrophic Lateral Sclerosis. Intriguingly, compelling evidence suggests a potential correlation in Fronto‐temporal dementia and Alzheimer's disease as well. Although the U snRNP biogenesis pathway is conserved across all eukaryotic cells, neurons, in particular, appear to be highly susceptible to alterations in spliceosome homeostasis. In contrast, other cell types exhibit a greater resilience to such disturbances. This vulnerability underscores the intricate relationship between U1 snRNP dynamics and the health of neuronal cells, shedding light on potential avenues for understanding and addressing neurodegenerative disorders.
... S5A). The binding pattern of U11-35K contrasts with that of its counterpart U1-70K (16), which only uses its RRM to bind SL1 of U1 snRNA (24,25). The ubiquitin-like domain-containing protein U11-25K simul-taneously interacts with SL1 to SL3 (Fig. 2, B and C, and fig. ...
... The central stem loop of U6atac snRNA is mainly stabilized by ZnF1 of CENATAC through H-bonds (Fig. 4G). At the center of this interface, Arg 24 donates two H-bonds to A52; Tyr 22 stacks against the nucleobase of U65. At the periphery, Lys 25 and Lys 110 contact the phosphates of G51 and G66, respectively. ...
The minor spliceosome, which is responsible for the splicing of U12-type introns, comprises five small nuclear RNAs (snRNAs), of which only one is shared with the major spliceosome. In this work, we report the 3.3-angstrom cryo–electron microscopy structure of the fully assembled human minor spliceosome pre-B complex. The atomic model includes U11 small nuclear ribonucleoprotein (snRNP), U12 snRNP, and U4atac/U6atac.U5 tri-snRNP. U11 snRNA is recognized by five U11-specific proteins (20K, 25K, 35K, 48K, and 59K) and the heptameric Sm ring. The 3′ half of the 5′-splice site forms a duplex with U11 snRNA; the 5′ half is recognized by U11-35K, U11-48K, and U11 snRNA. Two proteins, CENATAC and DIM2/TXNL4B, specifically associate with the minor tri-snRNP. A structural analysis uncovered how two conformationally similar tri-snRNPs are differentiated by the minor and major prespliceosomes for assembly.
... For the pre-A complex, the coordinates of human 17S U2 snRNP (this paper) and U1 snRNP (PDB 3CW1) 41 were docked into the EM maps for the U2 and U1 regions, respectively. For the U2 region, DNAJC8 was de novo built based on the EM map. ...
Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2–BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.
... The 5'-SS binding region of the U11 snRNA is not well resolved in the cryo-EM map, however, a continuous density can be traced from the bottom of the helix H pointing towards the outside of the Sm ring (Fig. 1). This is in agreement with the equivalent region in the U1 snRNP structure (33,36,37). ...
... Importantly, this specific recognition is transmitted to distal parts of the complex via a chain of interactions starting with SNRNP25, PDCD7 through SNRNP48 and ZMAT5 all the way to the proximity of the 5'SS binding region. There is a precedence for such a long-distance interaction in the U1 snRNP, where an extended tail of the U1-70K wraps around the Sm ring and contacts U1-C near the 5'SS (33,44). However, the components and the mechanism utilised by the U11 snRNP to achieve similar goals are very different. ...
The minor spliceosome catalyses the excision of U12-dependent introns from pre-mRNAs. These introns are rare, but their removal is critical for cell viability. We obtained a cryo-EM reconstruction of the 13-subunit U11 snRNP complex, revealing structures of U11 snRNA and five minor spliceosome-specific factors. U11 snRNP appears strikingly different from the equivalent major spliceosome U1 snRNP. SNRNP25 and SNRNP35 form a dimer, which specifically recognises U11 snRNA. PDCD7 forms extended helices, which bridge SNRNP25 and SNRNP48, located at the distal ends of the particle. SNRNP48 forms multiple interfaces with U11 snRNP and, together with ZMAT5, are positioned near the 5′-end of the U11 snRNA and likely stabilise the binding of the incoming 5′-SS. Our structure provides mechanistic insights into U12-dependent intron recognition and the evolution of the splicing machinery.
... SnRNAs have elaborate secondary and tertiary structures. The spatial organization of mature snRNAs in snRNPs and the spliceosome has been analyzed by chemical and enzymatic probing, and in recent years by X-ray crystallography and cryo-electron microscopy 3,[40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55] . In all reported snRNA structures, the Sm binding site is always singlestranded and occupied by Sm proteins. ...
... Then, we predicted suboptimal secondary structures for each pre-snRNA sequence using unconstrained prediction by UNAfold 58 with the exception of U1, to which we applied constrained prediction using RNAsubopt 59 . The unconstrained prediction by UNAfold did not provide consistent structures for U1 and we had to apply an additional constraint and blocked nucleotides (depicted by crosses in Fig. 1b) involved in the interaction with the SNRNP70 (U1-70K) protein from intramolecular base-pairing 52 . SNRNP70 was shown to interact with U1 pre-snRNA in the cytoplasm before or simultaneously with the SMN complex 60 . ...
... We therefore tested whether altering of the 3′ end extension affects folding of other pre-snRNAs, but we did not find any significant difference among structures with different lengths of 3′ end extra sequence for U2, U4, and U5 pre-snRNAs. In contrast, shortening of the 3′ extra sequence in U1 pre-snRNA sequence to only six extra nucleotides eliminated the compacted structure at the 3′ end and U1-pre-snRNA adopted a fold highly similar to the mature U1 snRNA structure, which indicates that U1 snRNA folding is specifically sensitive to 3′ extra sequence ( Fig. 1c and Fig. S5) 52,61 . We were unable to predict consistent common best representative structures for minor U11, U12, and U4atac pre-snRNAs. ...
Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3′-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.
... The U1 snRNA secondary structure consists of an unpaired 5′end, a four-way junction of 3 stem-loops (SL I-III) in a trefoil fold, a Sm site, and a fourth stem-loop at the 3′end (SL IV) (Figure 1b) [26]. This snRNA is transcribed in the nucleus by the RNA polymerase II (Figure 2a). ...
... However, not all base pairs at different 5′ss positions are equally important, and their contribution to splicing roughly correlates with their conservation. In eukaryotic organisms, in the 9 nucleotides consensus sequence (which can be expanded to include 11 base pairs), the most conserved 5′ss positions lie at the first two intronic positions (+1 and +2), which The U1 snRNA secondary structure consists of an unpaired 5 end, a four-way junction of 3 stem-loops (SL I-III) in a trefoil fold, a Sm site, and a fourth stem-loop at the 3 end (SL IV) (Figure 1b) [26]. This snRNA is transcribed in the nucleus by the RNA polymerase II (Figure 2a). ...
Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s—ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5′ splice-site (5′ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.
... with theoutput-countedmutations flag to tabulate mutation types observed at each sequence position. Area under the recei v er operating characteristic curves (AUROC) were calculated with Scikit-Learn (0.24.1) in Python using background-subtracted muta tion ra tes with pairing sta tus of each position deri v ed from known r efer ence structur es (41)(42)(43)(44)(45). To identify the G muta tion signa ture filter , A UROC was calculated for all possible combinations of m utation types, w hich revealed that including only G-to-C and G-to-T substitutions yielded the highest AUROC for all enzymes. ...
... The positi v e predicti v e value (ppv) and sensitivity (sens) of modeled structures were computed relati v e to accepted r efer ence structur es as pr eviously described ( 8 ), using all Watson Crick and GU pairs allowing for one-position register shifts and ignoring singleton pairs. Accepted r efer ence structur es wer e obtained from refs (41)(42)(43)(44)(45). Modifications to tmRNA and RMRP structur es wer e included as pr eviously described ( 8 ). ...
Chemical probing experiments have transformed RNA structure analysis, enabling high-throughput measurement of base-pairing in living cells. Dimethyl sulfate (DMS) is one of the most widely used structure probing reagents and has played a pivotal role in enabling next-generation single-molecule probing analyses. However, DMS has traditionally only been able to probe adenine and cytosine nucleobases. We previously showed that, using appropriate conditions, DMS can also be used to interrogate base-pairing of uracil and guanines in vitro at reduced accuracy. However, DMS remained unable to informatively probe guanines in cells. Here, we develop an improved DMS mutational profiling (MaP) strategy that leverages the unique mutational signature of N1-methylguanine DMS modifications to enable high-fidelity structure probing at all four nucleotides, including in cells. Using information theory, we show that four-base DMS reactivities convey greater structural information than current two-base DMS and SHAPE probing strategies. Four-base DMS experiments further enable improved direct base-pair detection by single-molecule PAIR analysis, and ultimately support RNA structure modeling at superior accuracy. Four-base DMS probing experiments are straightforward to perform and will broadly facilitate improved RNA structural analysis in living cells.
... First, in the nuclear extract (NE) prepared from HeLa cells, coimmunoprecipitation (co-IP) analysis using anti-U1C antibody showed that U1-70K and U1A, two other U1 snRNP-specific proteins, were significantly downregulated in the IPed sample using U1 AMO-treated NE (9). Second, it is well established that U1-70K can bind U1 snRNA stem loop 1 (SL1) (Fig. 1A) (10)(11)(12), which is close to the region where 25 nt U1 AMO binds, and U1C was also reported to be associated with U1 snRNA at the 5 0 end within U1 snRNP complex (13). Therefore, U1 AMO might interfere with the associations of U1 snRNA and U1-70K-U1C. ...
Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation (PCPA) of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP/RNAP polymerase II (RNAPII) interaction. By performing ChIP-seq for phosphorylation of Ser2 (Ser2P) and Ser5 (Ser5P) of the C-terminal domain (CTD) of RPB1, the largest subunit of RNAPII, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high Ser2P signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by ChIP-seq and iCLIP-seq analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.
... with the --output-counted-mutations flag to tabulate mutation types observed at each sequence position. Area under the receiver operating characteristic curves (AUROC) were calculated with Scikit-Learn (0.24.1) in Python using backgroundsubtracted mutation rates with pairing status of each position derived from known reference structures (41)(42)(43)(44)(45). To identify the G mutation signature filter, AUROC was calculated for all possible combinations of mutation types, which revealed that including only G-to-C and G-to-T substitutions yielded the highest AUROC for all enzymes. ...
... Quantification of model accuracy: The positive predictive value (ppv) and sensitivity (sens) of modeled structures were computed relative to accepted reference structures as previously described (8), using all Watson Crick and GU pairs allowing for one-position register shifts and ignoring singleton pairs. Accepted reference structures were obtained from refs (41)(42)(43)(44)(45). Modifications to tmRNA and RMRP structures were included as previously described (8). ...
Chemical probing experiments have transformed RNA structure analysis, enabling high-throughput measurement of base-pairing in living cells. Dimethyl sulfate (DMS) is one of the most widely used structure probing reagents and has played a prominent role in enabling next-generation single-molecule probing analyses. However, DMS has traditionally only been able to probe adenine and cytosine nucleobases. We previously showed that, using appropriate conditions, DMS can also be used to interrogate base-pairing of uracil and guanines in vitro at reduced accuracy. However, DMS remained unable to informatively probe guanines in cells. Here, we develop an improved DMS mutational profiling (MaP) strategy that leverages the unique mutational signature of N1-methylguanine DMS modifications to enable robust, high-fidelity structure probing at all four nucleotides, including in cells. Using information theory, we show that four-base DMS reactivities convey greater structural information than comparable two-base DMS and SHAPE probing strategies. Four-base DMS experiments further enable improved direct base-pair detection by single-molecule PAIR analysis, and ultimately support RNA structure modeling at superior accuracy. Four-base DMS probing experiments are easily performed and will broadly facilitate improved RNA structural analysis in living cells.
