The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

Structural insights into branch site proofreading by human spliceosome.

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.

Cellular nuclear-localized U2AF2 protein is hijacked by the flavivirus 3'UTR for viral replication complex formation and RNA synthesis.

Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3' untranslated region (3'UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3'UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3'UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3' UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.

SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns.

Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2 auxiliary factor (U2AF) heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. In vitro binding and nuclear magnetic resonance analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 small nuclear ribonucleoprotein (U2 snRNP) component in active spliceosomes. We propose a mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes.

Structural insights into recognition of SL4, the UUCG stem-loop, of human U1 snRNA by the ubiquitin-like domain, including the C-terminal tail in the SF3A1 subunit of U2 snRNP.

The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704-785) adopts a typical ß-grasp fold with a topology of ß1-ß2-α1-310a-ß3-ß4-310b-ß5, closely resembling that of ubiquitin, except for the length and structure of the ß1/ß2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (ß3), Phe763 (ß4) and Lys765 (following ß4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the ß-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding.

Phylogenetic Analysis of Spliceosome SF3a2 in Different Plant Species.

The formation of mature mRNA requires cutting introns and splicing exons. The occurrence of splicing involves the participation of the spliceosome. Common spliceosomes mainly include five snRNPs: U1, U2, U4/U6, and U5. SF3a2, an essential component of spliceosome U2 snRNP, participates in splicing a series of genes. There is no definition of SF3a2 in plants. The paper elaborated on SF3a2s from a series of plants through protein sequence similarity. We constructed the evolutionary relationship of SF3a2s in plants. Moreover, we analyzed the similarities and differences in gene structure, protein structure, the cis-element of the promoter, and expression pattern; we predicted their interacting proteins and constructed their collinearity. We have preliminarily analyzed SF3a2s in plants and clarified the evolutionary relationship between different species; these studies can better serve for in-depth research on the members of the spliceosome in plants.

Studying the connection between SF3B1 and four types of cancer by analyzing networks constructed based on published research.

Splicing factor 3B subunit 1 (SF3B1) is the largest component of SF3b protein complex which is involved in the pre-mRNA splicing mechanism. Somatic mutations of SF3B1 were shown to be associated with aberrant splicing, producing abnormal transcripts that drive cancer development and/or prognosis. In this study, we focus on the relationship between SF3B1 and four types of cancer, namely myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL) and breast cancer (BC). For this purpose, we identified from the Pubmed library only articles which mentioned SF3B1 in connection with the investigated types of cancer for the period 2007 to 2018 to reveal how the connection has developed over time. We left out all published articles which mentioned SF3B1 in other contexts. We retrieved the target articles and investigated the association between SF3B1 and the mentioned four types of cancer. For this we utilized some of the publicly available databases to retrieve gene/variant/disease information related to SF3B1. We used the outcome to derive and analyze a variety of complex networks that reflect the correlation between the considered diseases and variants associated with SF3B1. The results achieved based on the analyzed articles and reported in this article illustrated that SF3B1 is associated with hematologic malignancies, such as MDS, AML, and CLL more than BC. We found that different gene networks may be required for investigating the impact of mutant splicing factors on cancer development based on the target cancer type. Additionally, based on the literature analyzed in this study, we highlighted and summarized what other researchers have reported as the set of genes and cellular pathways that are affected by aberrant splicing in cancerous cells.

SART3 associates with a post-splicing complex.

SART3 is a multifunctional protein that acts in several steps of gene expression, including assembly and recycling of the spliceosomal U4/U6 small nuclear ribonucleoprotein particle (snRNP). In this work, we provide evidence that SART3 associates via its N-terminal HAT domain with the 12S U2 snRNP. Further analysis showed that SART3 associates with the post-splicing complex containing U2 and U5 snRNP components. In addition, we observed an interaction between SART3 and the RNA helicase DHX15, which disassembles post-splicing complexes. Based on our data, we propose a model that SART3 associates via its N-terminal HAT domain with the post-splicing complex, where it interacts with U6 snRNA to protect it and to initiate U6 snRNA recycling before a next round of splicing.

The role of DDX46 in breast cancer proliferation and invasiveness: A potential therapeutic target.

DDX46, a member of DEAD-box (DDX) proteins, is associated with various cancers, while its involvement in the pathogenesis of breast cancer hasn't been reported so far. The study demonstrated the overexpression of DDX46 in human breast cancer cells and tissue samples, and correlated with high histological grade and lymph node metastasis. Downregulation of DDX46 in the breast cancer cell lines inhibited their proliferation and invasiveness in vitro. Furthermore, the growth of MDA-MB-231 xenografts was suppressed in nude mice by DDX46 knockingdown. Taken together, our findings suggest that DDX46 is an oncogenic factor in human breast cancer, and a potential therapeutic target.

Phylogenetic analysis and stress response of the plant U2 small nuclear ribonucleoprotein B″ gene family.

BACKGROUND: Alternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein B″ (U2B″), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2B″, and its mechanism remains unclear. RESULT: Phylogenetic analysis, including gene and protein structures, revealed that U2B″ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2B″ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein-protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins. CONCLUSION: U2B″ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.

Splicing of branchpoint-distant exons is promoted by Cactin, Tls1 and the ubiquitin-fold-activated Sde2.

Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5'-splice site (5'ss), branchpoint (BP) and 3'-splice site (3'ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3'ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP-3'ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3'ss towards the spliceosome's catalytic centre by folding the RNA between the BP and 3'ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.

Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1.

SURP domains are exclusively found in splicing-related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1-α2-310 -α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α-helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16-residue α-helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio-layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 µM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.

Cancer-associated mutations in SF3B1 disrupt the interaction between SF3B1 and DDX42.

While cancer-associated SF3B1 mutations causes alternative RNA splicing, the molecular mechanism underlying the alternative RNA splicing is not fully elucidated. Here, we analysed the proteins that interacted with the wild-type and K700E-mutated SF3B1 and found that the interactions of two RNA helicases, DDX42 and DDX46, with the mutated SF3B1 were reduced. Overexpression of DDX42 restored the decreased interaction between DDX42 and the K700E-mutated SF3B1, and suppressed some alternative RNA splicing associated with the SF3B1 mutation. Mutation that decreased the ATP hydrolysis activities of DDX42 abolished the suppressive effects of DDX42 on the alternative RNA splicing, suggesting that the ATP hydrolysis activity of DDX42 is involved in the mechanism of the altered RNA splicing associated with the SF3B1 mutation. Our study demonstrates an important function of the interaction between DDX42 and SF3B1 on regulating RNA splicing and revealed a potential role of DDX42 in the altered RNA splicing associated with the SF3B1 mutation.

An Arabidopsis Retention and Splicing complex regulates root and embryo development through pre-mRNA splicing.

Pre-mRNA splicing is an important step in the posttranscriptional processing of transcripts and a key regulator of development. The heterotrimeric retention and splicing (RES) complex plays vital roles in the growth and development of yeast, zebrafish, and humans by mediating pre-mRNA splicing of multiple genes. However, whether the RES complex is conserved in plants and what specific functions it has remain unknown. In this study, we identified Arabidopsis (Arabidopsis thaliana) BUD13 (AtBUD13), GROWTH, DEVELOPMENT AND SPLICING 1 (GDS1), and DAWDLE (DDL) as the counterparts of the yeast RES complex subunits Bud site selection protein 13 (Bud13), U2 snRNP component Snu17 (Snu17), and Pre-mRNA leakage protein 1, respectively. Moreover, we showed that RES is an ancient complex evolutionarily conserved in eukaryotes. GDS1 directly interacts with both AtBUD13 and DDL in nuclear speckles. The BUD13 domain of AtBUD13 and the RNA recognition motif domain of GDS1 are necessary and sufficient for AtBUD13-GDS1 interaction. Mutants of AtBUD13, GDS1, and DDL failed to properly splice multiple genes involved in cell proliferation and showed defects in early embryogenesis and root development. In addition, we found that GDS1 and DDL interact, respectively, with the U2 small nuclear ribonucleoproteins auxiliary factor AtU2AF65B and the NineTeen Complex-related splicing factor SKIP, which are essential for early steps of spliceosome assembly and recognition of splice sites. Altogether, our work reveals that the Arabidopsis RES complex is important for root and early embryo development by modulating pre-mRNA splicing.

Sequence-specific RNA recognition by an RGG motif connects U1 and U2 snRNP for spliceosome assembly.

In mammals, the structural basis for the interaction between U1 and U2 small nuclear ribonucleoproteins (snRNPs) during the early steps of splicing is still elusive. The binding of the ubiquitin-like (UBL) domain of SF3A1 to the stem-loop 4 of U1 snRNP (U1-SL4) contributes to this interaction. Here, we determined the 3D structure of the complex between the UBL of SF3A1 and U1-SL4 RNA. Our crystallography, NMR spectroscopy, and cross-linking mass spectrometry data show that SF3A1-UBL recognizes, sequence specifically, the GCG/CGC RNA stem and the apical UUCG tetraloop of U1-SL4. In vitro and in vivo mutational analyses support the observed intermolecular contacts and demonstrate that the carboxyl-terminal arginine-glycine-glycine-arginine (RGGR) motif of SF3A1-UBL binds sequence specifically by inserting into the RNA major groove. Thus, the characterization of the SF3A1-UBL/U1-SL4 complex expands the repertoire of RNA binding domains and reveals the capacity of RGG/RG motifs to bind RNA in a sequence-specific manner.

Phylogeny and conservation of plant U2A/U2A', a core splicing component in U2 spliceosomal complex.

MAIN CONCLUSION: This study systematically identifies 112 U2A genes from 80 plant species by combinatory bioinformatics analysis, which is important for understanding their phylogenetic history, expression profiles and for predicting specific functions. In eukaryotes, a pre-mRNA can generate multiple transcripts by removing certain introns and joining corresponding exons, thus greatly expanding the transcriptome and proteome diversity. The spliceosome is a mega-Dalton ribonucleoprotein (RNP) complex that is essential for the process of splicing. In spliceosome components, the U2 small nuclear ribonucleoprotein (U2 snRNP) forms the pre-spliceosome by association with the branch site. An essential component that promotes U2 snRNP assembly, named U2A, has been extensively identified in humans, yeast and nematodes. However, studies examining U2A genes in plants are scarce. In this study, we performed a comprehensive analysis and identified a total of 112 U2A genes from 80 plant species representing dicots, monocots, mosses and algae. Comparisons of the gene structures, protein domains, and expression patterns of 112 U2A genes indicated that the conserved functions were likely retained by plant U2A genes and important for responses to internal and external stimuli. In addition, analysis of alternative transcripts and splice sites of U2A genes indicated that the fifth intron contained a conserved alternative splicing event that might be important for its molecular function. Our work provides a general understanding of this splicing factor family in terms of genes and proteins, and it will serve as a fundamental resource that will contribute to further mechanistic characterization in plants.

Integrated Bioinformatics Analysis Reveals Marker Genes and Potential Therapeutic Targets for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a rare cardiovascular disease with very high mortality rate. The currently available therapeutic strategies, which improve symptoms, cannot fundamentally reverse the condition. Thus, new therapeutic strategies need to be established. Our research analyzed three microarray datasets of lung tissues from human PAH samples retrieved from the Gene Expression Omnibus (GEO) database. We combined two datasets for subsequent analyses, with the batch effects removed. In the merged dataset, 542 DEGs were identified and the key module relevant to PAH was selected using WGCNA. GO and KEGG analyses of DEGs and the key module indicated that the pre-ribosome, ribosome biogenesis, centriole, ATPase activity, helicase activity, hypertrophic cardiomyopathy, melanoma, and dilated cardiomyopathy pathways are involved in PAH. With the filtering standard (|MM| > 0.95 and |GS| > 0.90), 70 hub genes were identified. Subsequently, five candidate marker genes (CDC5L, AP3B1, ZFYVE16, DDX46, and PHAX) in the key module were found through overlapping with the top thirty genes calculated by two different methods in CytoHubb. Two of them (CDC5L and DDX46) were found to be significantly upregulated both in the merged dataset and the validating dataset in PAH patients. Meanwhile, expression of the selected genes in lung from PAH chicken measured by qRT-PCR and the ROC curve analyses further verified the potential marker genes' predictive value for PAH. In conclusion, CDC5L and DDX46 may be marker genes and potential therapeutic targets for PAH.

SPF45/RBM17-dependent, but not U2AF-dependent, splicing in a distinct subset of human short introns.

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.

Inactivation of the tumor suppressor p53 by long noncoding RNA RMRP.

p53 inactivation is highly associated with tumorigenesis and drug resistance. Here, we identify a long noncoding RNA, the RNA component of mitochondrial RNA-processing endoribonuclease (RMRP), as an inhibitor of p53. RMRP is overexpressed and associated with an unfavorable prognosis in colorectal cancer. Ectopic RMRP suppresses p53 activity by promoting MDM2-induced p53 ubiquitination and degradation, while depletion of RMRP activates the p53 pathway. RMRP also promotes colorectal cancer growth and proliferation in a p53-dependent fashion in vitro and in vivo. This anti-p53 action of RMRP is executed through an identified partner protein, SNRPA1. RMRP can interact with SNRPA1 and sequester it in the nucleus, consequently blocking its lysosomal proteolysis via chaperone-mediated autophagy. The nuclear SNRPA1 then interacts with p53 and enhances MDM2-induced proteasomal degradation of p53. Remarkably, ablation of SNRPA1 completely abrogates RMRP regulation of p53 and tumor cell growth, indicating that SNRPA1 is indispensable for the anti-p53 function of RMRP. Interestingly and significantly, poly (ADP-ribose) polymerase (PARP) inhibitors induce RMRP expression through the transcription factor C/EBPß, and RMRP confers tumor resistance to PARP inhibition by preventing p53 activation. Altogether, our study demonstrates that RMRP plays an oncogenic role by inactivating p53 via SNRPA1 in colorectal cancer.

A prometastatic splicing program regulated by SNRPA1 interactions with structured RNA elements.

Aberrant alternative splicing is a hallmark of cancer, yet the underlying regulatory programs that control this process remain largely unknown. Here, we report a systematic effort to decipher the RNA structural code that shapes pathological splicing during breast cancer metastasis. We discovered a previously unknown structural splicing enhancer that is enriched near cassette exons with increased inclusion in highly metastatic cells. We show that the spliceosomal protein small nuclear ribonucleoprotein polypeptide A' (SNRPA1) interacts with these enhancers to promote cassette exon inclusion. This interaction enhances metastatic lung colonization and cancer cell invasion, in part through SNRPA1-mediated regulation of PLEC alternative splicing, which can be counteracted by splicing modulating morpholinos. Our findings establish a noncanonical regulatory role for SNRPA1 as a prometastatic splicing enhancer in breast cancer.