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PRP5 and TAT-SF1 are located near the BSL and interact with SF3B1HEAT a, Location of the PRP5 RecA1 and RecA2 domains, and interaction of the PRP5 α-helix with SF3B1HEAT, and TAT-SF1 RRM1 and UHM with SF3B1HEAT and the BSL. Dotted line denotes the potential path of unstructured PRP5 regions, based on crosslinking data. b, Fit of TAT-SF1RRM1 into cryo-EM density adjacent to SF3B1 HR15–HR16. A density element that could not be unambiguously defined, but is probably part of TAT-SF1, contacts the BSL loop. c, The BSL is sequestered by PRP5, TAT-SF1, SF3B1 and the unassigned protein region (UPR). Grey denotes cryo-EM density of the unassigned protein region. Coloured surfaces are derived from fitted protein models.
Source publication
The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing¹. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52,3,4,5,6,7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs...
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Proper follicle development is very important for the production of mature oocytes, which is essential for the maintenance of female fertility. This complex biological process requires precise gene regulation. The most abundant modification of mRNA, N6-methyladenosine (m6A), is involved in many RNA metabolism processes, including RNA splicing, tran...
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. ...
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.
... The catalytic center is in the middle of the RNA helix. (b) The human 17S U2 snRNP involved in the first step of RNA splicing [98], showing the RNA core and the protein most closely associated with it (splicing factor 3b). Again, the part of the protein in contact with the RNA is lacking in regular secondary structure. ...
Darwin’s theory of evolution by natural selection was revolutionary because it provided a mechanism by which variation could be selected. This mechanism can only operate on living systems and thus cannot be applied to the origin of life. Here, we propose a viable alternative mechanism for prebiotic systems: autocatalytic selection, in which molecules catalyze reactions and processes that lead to increases in their concentration. Crucially, this provides a driver for increases in concentrations of molecules to a level that permits prebiotic metabolism. We show how this can produce high levels of amino acids, sugar phosphates, nucleotides and lipids and then lead on to polymers. Our outline is supported by a set of guidelines to support the identification of the most likely prebiotic routes. Most of the steps in this pathway are already supported by experimental results. These proposals generate a coherent and viable set of pathways that run from established Hadean geochemistry to the beginning of life.
... The human ID spliceosome prior to activation has been structurally characterized in atomic details. [6][7][8][9][10][11][12][13][14] U1 and U2 small nuclear ribonucleoproteins (snRNPs) recognize the 5′-splice site (5′ SS) and the branch point sequence (BPS), respectively, to form the pre-spliceosome (known as the A complex). [15][16][17] The A complex associates with U4/U6.U5 tri-snRNP to form the pre-B complex, in which U2 snRNP is connected to tri-snRNP mainly through the U2/ U6 duplex. ...
Spliceosome is often assembled across an exon and undergoes rearrangement to span a neighboring intron. Most states of the intron-defined spliceosome have been structurally characterized. However, the structure of a fully assembled exon-defined spliceosome remains at large. During spliceosome assembly, the pre-catalytic state (B complex) is converted from its precursor (pre-B complex). Here we report atomic structures of the exon-defined human spliceosome in four sequential states: mature pre-B, late pre-B, early B, and mature B. In the previously unknown late pre-B state, U1 snRNP is already released but the remaining proteins are still in the pre-B state; unexpectedly, the RNAs are in the B state, with U6 snRNA forming a duplex with 5′-splice site and U5 snRNA recognizing the 3′-end of the exon. In the early and mature B complexes, the B-specific factors are stepwise recruited and specifically recognize the exon 3′-region. Our study reveals key insights into the assembly of the exon-defined spliceosomes and identifies mechanistic steps of the pre-B-to-B transition.
... Structural analysis revealed that these dense protein-RNA interactions could be involved in constraining the length of the U2/intron duplex to be 16 bps (−13 nt to +2 nt around BP) [51][52][53][54] . Importantly, the −13 nt is located at the 5' end of the U2/intron duplex, where U2 snRNA starts to form stem loop IIa (SLIIa) onward 62,63 . Thus, protein-RNA and RNA-RNA interactions at this location could potentially affect U2 snRNP/intron engagement and RNA splicing. ...
Protein arginine methyltransferase 9 (PRMT9) is a recently identified member of the PRMT family, yet its biological function remains largely unknown. Here, by characterizing an intellectual disability associated PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncover an important function of PRMT9 in neuronal development. The G189R mutation abolishes PRMT9 methyltransferase activity and reduces its protein stability. Knockout of Prmt9 in hippocampal neurons causes alternative splicing of ~1900 genes, which likely accounts for the aberrant synapse development and impaired learning and memory in the Prmt9 cKO mice. Mechanistically, we discover a methylation-sensitive protein–RNA interaction between the arginine 508 (R508) of the splicing factor 3B subunit 2 (SF3B2), the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element that is critical for RNA splicing. Additionally, using human and mouse cell lines, as well as an SF3B2 arginine methylation-deficient mouse model, we provide strong evidence that SF3B2 is the primary methylation substrate of PRMT9, thus highlighting the conserved function of the PRMT9/SF3B2 axis in regulating pre-mRNA splicing.
... One 491 possible explanation is presence or absence of additional cofactors that coordinate splicing with mutant 492 spliceosome; For example, mutation on SF3B1 attenuates recruitment of SUGP1, a G-PATCH protein, that 493 recruits DHX15 to surveil and quality control splicing under suboptimal conditions thereby potentiating cryptic 494 splicing 55,56 . Advances in cryo-EM technologies have started to elucidate the dynamics of the early splicing where 495 faithful branchpoint selection is made through several high-resolution structures of early spliceosome [57][58][59][60] . ...
SF3B1 is the most recurrently mutated RNA splicing factor in cancer; However, its study has been hindered by a lack of disease-relevant cell line models. Here, we compared four genome engineering platforms to establish SF3B1 mutant cell lines: CRISPR-Cas9 editing, AAV HDR editing, base editing (ABEmax, ABE8e), and prime editing (PE2, PE3, PE5Max). We showed that prime editing via PE5max achieved the most efficient SF3B1 K700E editing across a wide range of cell lines. We further refined our approach by coupling prime editing with a with a fluorescent reporter that leverages a SF3B1 mutation-responsive synthetic intron to mark prime edited cells. Calling this approach prime editing coupled intron-assisted selection (PRECIS), we then introduced the K700E hotspot mutation into two chronic lymphocytic leukemia (CLL) cell lines, HG-3 and MEC-1, and demonstrated that our PRECIS-engineered cells faithfully recapitulate the altered splicing and copy number variation (CNV) events frequently found in CLL patients with SF3B1 mutation. Our results showcase PRECIS as an efficient and generalizable method for engineering genetically faithful SF3B1 mutant models, shed new light on the role of SF3B1 mutation in cancer biology, and enables generation of novel SF3B1 mutant cell lines in any cellular context.
... The RNA helicase PRP5 (also known as DDX46) plays a key role during the assembly of the A complex [4][5][6][12][13][14][15] . In human 17S U2 snRNP, U2 snRNA is unable to pair up with the BS because the relevant sequences of U2 snRNA form the double-stranded BSL 16,17 . ATP hydrolysis by PRP5 leads to the unwinding of BSL, thus promoting the formation of the U2-BS duplex. ...
... The 2.5 Å EM map reveals and allows atomic modeling of the interface between PRP5 and SF3B1 (Fig. 2a), which has remained elusive despite several published structures of human 17S U2 snRNP 16,32,34 . Three structural motifs at the N-terminal half of PRP5 closely interact with SF3B1, tethering PRP5 to U2 snRNP. ...
... The 5′ sequence of U2 snRNA in human 17S U2 snRNP comprises three elements: stem-loop I (SLI, nucleotides G12 through C21), BSL (U25 through C45) and stem-loop IIa (SLIIa, U46 through U65) 16,17 (Fig. 3a). The three-way junction of U2 snRNA is stabilized by SF3A3, SF3B2, SF3B1, TAT-SF1 and PRP5. ...
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.
... There are now multiple cryo-electron microscopy (cryo-EM) structures of human spliceosomal complexes: pre-A, pre-B, B, B act , C, C * , P, and ILS, as well as the 17S U2 snRNP (the major subunit of E and A complexes) Bertram et al. 2017a,b;Zhang et al. 2017Zhang et al. , 2018Zhang et al. , 2019bZhang et al. , 2020Zhang et al. , 2022bHaselbach et al. 2018;; Tholen et al. 2022). However, SUGP1 was not observed in any of these stage-specific complexes, suggesting that SUGP1 likely participates in an uncharacterized intermediate complex during branch site recognition and that its interactions are expected to be transient. ...
The spliceosomal gene SF3B1 is frequently mutated in cancer. While it is known that SF3B1 hotspot mutations lead to loss of splicing factor SUGP1 from spliceosomes, the cancer-relevant SF3B1–SUGP1 interaction has not been characterized. To address this issue, we show by structural modeling that two regions flanking the SUGP1 G-patch make numerous contacts with the region of SF3B1 harboring hotspot mutations. Experiments confirmed that all the cancer-associated mutations in these regions, as well as mutations affecting other residues in the SF3B1–SUGP1 interface, not only weaken or disrupt the interaction but also alter splicing similarly to SF3B1 cancer mutations. Finally, structural modeling of a trimeric protein complex reveals that the SF3B1–SUGP1 interaction “loops out” the G-patch for interaction with the helicase DHX15. Our study thus provides an unprecedented molecular view of a protein complex essential for accurate splicing and also reveals that numerous cancer-associated mutations disrupt the critical SF3B1–SUGP1 interaction.
... The molecular events leading to stable association of the U2 snRNP with the intron BP are only partially understood. The E-complex, containing pre-mRNA and U1 snRNP Reed 1993, 1991;Li et al. 2019), must merge with a form of the U2 snRNP carrying the SF3a and SF3b proteins (called 17S U2 snRNP in human, Brosi et al. 1993bBrosi et al. , 1993a to recognize the BP and form the prespliceosome or A-complex, within which the 5'ss and BP are base paired to snRNAs U1 and U2, respectively (Zhang et al. 2020b;Plaschka et al. 2018). As the prespliceosome forms, proteins present in E-complex and the U2 snRNP are released to allow new RNA-RNA interactions to be established. ...
Intron branch point (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity.
To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeast Saccharomyces cerevisiae by changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Splicing is inhibited within minutes after addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during co-transcriptional splicing in Plad-B using single-molecule intron tracking (SMIT) to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.
... Studies using the S. cerevisiae (yeast) homolog, Hsh155, show that MDS mutations change how the splicing machinery utilizes weak branch sites that contain mismatches between the intron and U2 snRNA as well as change interactions between Hsh155 and a conserved DEAD-box ATPase (Prp5/DDX46) that regulates the process [60,61]. In fact, cryo-EM structures of the human U2 snRNP show that the N-terminal region of Prp5/DDX46 nearly encircles SF3B1 and binds near the MDS mutation hotspot [62]. ...
Precursor mRNA (pre-mRNA) splicing is an essential step in human gene expression and is carried out by a large macromolecular machine called the spliceosome. Given the spliceosome's role in shaping the cellular transcriptome, it is not surprising that mutations in the splicing machinery can result in a range of human diseases and disorders (spliceosomopathies). This review serves as an introduction into the main features of the pre-mRNA splicing machinery in humans and how changes in the function of its components can lead to diseases ranging from blindness to cancers. Recently, several drugs have been developed that interact directly with this machinery to change splicing outcomes at either the single gene or transcriptome-scale. We discuss the mechanism of action of several drugs that perturb splicing in unique ways. Finally, we speculate on what the future may hold in the emerging area of spliceosomopathies and spliceosome-targeted treatments.
... Mutations in U2AF1 may affect the binding a nity of the protein to RNA, leading to aberrant splicing patterns and contributing to the development of various diseases, including myelodysplastic syndromes (50,51) and lung cancer (52). The SF3b complex is an essential part of the spliceosome and is a predominant component of U2 and U11/U12 snRNPs (53). It plays a crucial role in recognizing the branch-point sequence (BPS), stabilizing the U2snRNA/BPS duplex, and preventing any untimely transesteri cation (54). ...
Background
RNA binding proteins (RBPs), especially cell-specific RBPs are involved in critical processes such as alternative splicing of messenger RNAs and translational control, leading to the expression of cell-specific functional proteins. However, the expression pattern of RBPs in different cells of rheumatoid arthritis and their associated aberrant regulation remain largely unexplored.
Methods
We collected 2141 RNA binding protein genes (RBPs) from literature and identified cell populations present in rheumatoid arthritis and osteoarthritis control samples using single-cell data. We compared the changes in the relative proportions of cell classes between them and analyzed RBP expression patterns specific to different cell types. We investigated fibroblast cell populations and their cellular communication with different immune cells. Additionally, we used bulk RNA-seq data from rheumatoid arthritis and osteoarthritis samples to identify highly conserved variable splicing events and established a co-variation network of RBPs and these splicing events.
Results
We observed a greater number of down-regulated RBPs in each cell type, except for fibroblasts, endothelial cells, and macrophages, where the number of up-regulated genes was much higher. In fibroblasts from RA and OA patients, we identified 105 upregulated RBPs and 133 downregulated RBPs. These RBPs were co-expressed with genes enriched in various functional pathways, including extracellular matrix organization, cell adhesion, collagen fibril organization, and cytokine signaling. Cellular communication analysis demonstrated enhanced signaling pathways, like CXCL12-CXCR4, between fibroblasts and macrophages in RA. We identified a total of 715 differentially variable splicing events in our study, and alternative 5' and 3' splicing were the most prevalent. Some RBPs, such as MBNL2 in endothelial cells and U2AF1, SF3B6, and SF3B14 in fibroblast cells, may play a role in the pathogenesis of RA through splicing regulation.
Conclusion
In this study, we analyzed single-cell datasets to identify the inherent characteristics and abnormal expression patterns of RBPs in different cell types of patients with RA. Our findings revealed that certain cell-specific RBPs were associated with inflammatory signaling pathways and splicing regulation in RA. These findings suggest that the dysregulation of RBPs may contribute to the development of RA and highlight potential pathways for therapeutic interventions.
