The U1-70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to ß-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing.

Expression and Purification of Human U1-70K (snRNP70) and its BAD Domains Using an E. coli Expression System.

U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.

Functional analysis of the zinc finger modules of the Saccharomyces cerevisiae splicing factor Luc7.

Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28.

Systemic correlates of cutaneous manifestations of lupus erythematosus.

BACKGROUND: We aimed to investigate the prevalence of skin disease among patients with systemic lupus erythematosus (SLE) and determine whether LE skin disease had clinical or serologic correlates with SLE. METHODS: We reviewed records of 335 patients with SLE (seen at Mayo Clinic, Rochester, Minnesota, USA) and abstracted skin manifestations, fulfilled mucocutaneous SLE criteria, and clinical and serologic parameters. RESULTS: Of the 231 patients with skin manifestations, 57 (24.7%) had LE-specific conditions, 102 (44.2%) had LE-nonspecific conditions, and 72 (31.2%) had both. LE skin disease was associated with photosensitivity, anti-Smith antibodies, and anti-U1RNP antibodies (all P < 0.001). Patients without LE skin disease more commonly had elevated C-reactive protein levels (P = 0.01). Patients meeting 2-4 mucocutaneous American College of Rheumatology criteria less commonly had cytopenia (P = 0.004) or anti-double-stranded DNA antibodies (P = 0.004). No significant associations were observed for systemic involvement (renal, hematologic, neurologic, and arthritis) when comparing patients with or without LE skin involvement. LE skin involvement was not significantly associated with internal SLE disease flare, number of medications, or overall survival. CONCLUSIONS: LE skin disease commonly occurs in patients with SLE. The presence of LE skin disease had no mitigating impact on the severity of SLE sequelae, disease flares, number of medications, or overall survival.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast.

The recognition of the 5' splice site (5' ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5' ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5' ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5' ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

Anti-U1RNP antibodies are associated with a distinct clinical phenotype and a worse survival in patients with systemic sclerosis.

OBJECTIVES: To clarify the impact of anti-U1RNP antibodies on the clinical features and prognosis of patients with SSc. METHODS: We conducted a monocentric case-control, retrospective, longitudinal study. For each patient with SSc and anti-U1RNP antibodies (SSc-RNP+), one patient with mixed connective tissue disease (MCTD) and 2 SSc patients without anti-U1RNP antibodies (SSc-RNP-) were matched for age, sex, and date of inclusion. RESULTS: Sixty-four SSc-RNP+ patients were compared to 128 SSc-RNP- and 64 MCTD patients. Compared to SSc-RNP-, SSc-RNP+ patients were more often of Afro-Caribbean origin (31.3% vs. 11%, p < 0.01), and more often had an overlap syndrome than SSc-RNP- patients (53.1 % vs. 22.7%, p < 0.0001), overlapping with Sjögren's syndrome (n = 23, 35.9%) and/or systemic lupus erythematosus (n = 19, 29.7%). SSc-RNP+ patients were distinctly different from MCTD patients but less often had joint involvement (p < 0.01). SSc-RNP+ patients more frequently developed interstitial lung disease (ILD) (73.4% vs. 55.5% vs. 31.3%, p < 0.05), pulmonary fibrosis (PF) (60.9% vs. 37.5% vs. 10.9%, p < 0.0001), SSc associated myopathy (29.7% vs. 6.3% vs. 7.8%, p < 0.0001), and kidney involvement (10.9% vs. 2.3% vs. 1.6%, p < 0.05). Over a 200-month follow-up period, SSc-RNP+ patients had worse overall survival (p < 0.05), worse survival without PF occurrence (p < 0.01), ILD or PF progression (p < 0.01 and p < 0.0001). CONCLUSIONS: In SSc patients, anti-U1RNP antibodies are associated with a higher incidence of overlap syndrome, a distinct clinical phenotype, and poorer survival compared to SSc-RNP- and MCTD patients. Our study suggests that SSc-RNP+ patients should be separated from MCTD patients and may constitute an enriched population for progressive lung disease.

Structural insights into human exon-defined spliceosome prior to activation.

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.

Evolution of diagnostic criteria and new insights into clinical testing in mixed connective tissue disease; anti-survival motor neuron complex antibody as a novel marker of severity of the disease.

Mixed connective tissue disease (MCTD) is an autoimmune disorder characterized by a combination of clinical features from systemic lupus erythematosus, systemic sclerosis, and inflammatory muscle disease, along with the presence of positive anti-U1-ribonucleoprotein (U1-RNP) antibodies. The exact etiology of the disease remains unclear, but it is believed to involve vascular damage within the context of heightened autoimmune responses. Consequently, Raynaud's phenomenon and pulmonary arterial hypertension are observed in patients with MCTD. While specific biomarkers for MCTD have not yet been identified, the recent study of the utility of anti-survival motor neuron complex (SMN) antibodies in MCTD suggests a promising avenue for further research and the accumulation of additional evidence.

Clinical significance of small nuclear ribonucleoprotein U1 subunit 70 in patients with hepatocellular carcinoma.

Background & Aims: Small nuclear ribonucleoprotein U1 subunit 70 (SNRNP70) as one of the components of the U1 small nuclear ribonucleoprotein (snRNP) is rarely reported in cancers. This study aims to estimate the application potential of SNRNP70 in hepatocellular carcinoma (HCC) clinical practice. Methods: Based on the TCGA database and cohort of HCC patients, we investigated the expression patterns and prognostic value of SNRNP70 in HCC. Then, the combination of SNRNP70 and alpha-fetoprotein (AFP) in 278 HCC cases was analyzed. Next, western blotting and immunohistochemistry were used to detect the expression of SNRNP70 in nucleus and cytoplasm. Finally, Cell Counting Kit-8 (CCK-8) and scratch wound healing assays were used to detect the effect of SNRNP70 on the proliferation and migration of HCC cells. Results: SNRNP70 was highly expressed in HCC. Its expression was increasingly high during the progression of HCC and was positively related to immune infiltration cells. Higher SNRNP70 expression indicated a poor outcome of HCC patients. In addition, nuclear SNRNP70/AFP combination could be a prognostic biomarker for overall survival and recurrence. Cell experiments confirmed that knockdown of SNRNP70 inhibited the proliferation and migration of HCC cells. Conclusion: SNRNP70 may be a new biomarker for HCC progression and HCC diagnosis as well as prognosis. SNRNP70 combined with serum AFP may indicate the prognosis and recurrence status of HCC patients after operation.

U1 snRNP Biogenesis Defects in Neurodegenerative Diseases.

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.

Transcriptome and proteomic analysis of mpox virus F3L-expressing cells.

Background: Monkeypox or mpox virus (mpox) is a double-stranded DNA virus that poses a significant threat to global public health security. The F3 protein, encoded by mpox, is an apoenzyme believed to possess a double-stranded RNA-binding domain (dsRBD). However, limited research has been conducted on its function. In this study, we present data on the transcriptomics and proteomics of F3L-transfected HEK293T cells, aiming to enhance our comprehension of F3L. Methods: The gene expression profiles of pCAGGS-HA-F3L transfected HEK293T cells were analyzed using RNA-seq. Proteomics was used to identify and study proteins that interact with F3L. Real-time PCR was used to detect mRNA levels of several differentially expressed genes (DEGs) in HEK293T cells (or Vero cells) after the expression of F3 protein. Results: A total of 14,822 genes were obtained in cells by RNA-Seq and 1,672 DEGs were identified, including 1,156 up-regulated genes and 516 down-regulated genes. A total of 27 cellular proteins interacting with F3 proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and 19 cellular proteins with large differences in abundance ratios were considered to be candidate cellular proteins. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the DEGs were significantly enriched in immune-related pathways, including type I interferon signaling pathway, response to virus, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, etc. Moreover, some selected DEGs were further confirmed by real-time PCR and the results were consistent with the transcriptome data. Proteomics data show that cellular proteins interacting with F3 proteins are mainly related to RNA splicing and protein translation. Conclusions: Our analysis of transcriptomic and proteomic data showed that (1) F3L up-regulates the transcript levels of key genes in the innate immune signaling pathway, such as RIGI, MDA5, IRF5, IRF7, IRF9, ISG15, IFNA14, and elicits a broad spectrum of antiviral immune responses in the host. F3L also increases the expression of the FOS and JNK genes while decreasing the expression of TNFR2, these factors may ultimately induce apoptosis. (2) F3 protein interacts with host proteins involved in RNA splicing and protein translation, such as SNRNP70, POLR2H, HNRNPA1, DDX17, etc. The findings of this study shed light on the function of the F3 protein.

Engineering U1-Based Tetracycline-Inducible Riboswitches to Control Gene Expression in Mammals.

Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.

Stress-induced nuclear speckle reorganization is linked to activation of immediate early gene splicing.

Current models posit that nuclear speckles (NSs) serve as reservoirs of splicing factors and facilitate posttranscriptional mRNA processing. Here, we discovered that ribotoxic stress induces a profound reorganization of NSs with enhanced recruitment of factors required for splice-site recognition, including the RNA-binding protein TIAR, U1 snRNP proteins and U2-associated factor 65, as well as serine 2 phosphorylated RNA polymerase II. NS reorganization relies on the stress-activated p38 mitogen-activated protein kinase (MAPK) pathway and coincides with splicing activation of both pre-existing and newly synthesized pre-mRNAs. In particular, ribotoxic stress causes targeted excision of retained introns from pre-mRNAs of immediate early genes (IEGs), whose transcription is induced during the stress response. Importantly, enhanced splicing of the IEGs ZFP36 and FOS is accompanied by relocalization of the corresponding nuclear mRNA foci to NSs. Our study reveals NSs as a dynamic compartment that is remodeled under stress conditions, whereby NSs appear to become sites of IEG transcription and efficient cotranscriptional splicing.

Doubtful Clinical Value of Subtyping Anti-U1-RNP Antibodies Regarding the RNP-70 kDa Antigen in Sera of Patients with Systemic Lupus Erythematosus.

The detection of antinuclear antibodies is central to the diagnosis and prognosis of systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS) and mixed connective tissue disease (MCTD). Anti-U1-RNP and anti-RNP70 antibodies were assayed in the sera of patients with SLE (n = 114), pSS (n = 54) and MCTD (n = 12). In the SLE group, 34/114 (30%) were anti-U1-RNP positive, and 21/114 (18%) were both anti-RNP70 positive and anti-U1-RNP positive. In the MCTD group, 10/12 (83%) were anti-U1-RNP positive, and 9/12 (75%) were anti-RNP70 positive. Only one individual with pSS was antibody positive (for both anti-U1-RNP and anti-RNP70). All anti-RNP70-positive samples were also anti-U1-RNP positive. Anti-U1-RNP-positive subjects with SLE were younger (p < 0.0001); showed lower concentrations of complement protein 3 (p = 0.03); had lower eosinophil (p = 0.0005), lymphocyte (p = 0.006) and monocyte (p = 0.03) counts; and had accrued less organ damage (p = 0.006) than the anti-U1-RNP-negative SLE patients. However, we observed no significant clinical or laboratory parameter differences between the anti-U1-RNP-positive individuals with/without anti-RNP70 in the SLE group. In conclusion, anti-RNP70 antibodies are not exclusive to MCTD but are rarely detected in pSS and healthy individuals. In SLE, anti-U1-RNP antibodies are associated with a clinical phenotype that resembles MCTD, with hematologic involvement and less damage accrual. Based on our results, the clinical value of subtyping anti-RNP70 in anti-U1-RNP-positive sera appears to be of limited value.

Dissecting Detergent-Insoluble Proteome in Alzheimer's Disease by TMTc-Corrected Quantitative Mass Spectrometry.

Protein aggregation of amyloid-ß peptides and tau are pathological hallmarks of Alzheimer's disease (AD), which are often resistant to detergent extraction and thus enriched in the insoluble proteome. However, additional proteins that coaccumulate in the detergent-insoluble AD brain proteome remain understudied. Here, we comprehensively characterized key proteins and pathways in the detergent-insoluble proteome from human AD brain samples using differential extraction, tandem mass tag (TMT) labeling, and two-dimensional LC-tandem mass spectrometry. To improve quantification accuracy of the TMT method, we developed a complement TMT-based strategy to correct for ratio compression. Through the meta-analysis of two independent detergent-insoluble AD proteome datasets (8914 and 8917 proteins), we identified 190 differentially expressed proteins in AD compared with control brains, highlighting the pathways of amyloid cascade, RNA splicing, endocytosis/exocytosis, protein degradation, and synaptic activity. To differentiate the truly detergent-insoluble proteins from copurified background during protein extraction, we analyzed the fold of enrichment for each protein by comparing the detergent-insoluble proteome with the whole proteome from the same AD samples. Among the 190 differentially expressed proteins, 84 (51%) proteins of the upregulated proteins (n = 165) were enriched in the insoluble proteome, whereas all downregulated proteins (n = 25) were not enriched, indicating that they were copurified components. The vast majority of these enriched 84 proteins harbor low-complexity regions in their sequences, including amyloid-ß, Tau, TARDBP/TAR DNA-binding protein 43, SNRNP70/U1-70K, MDK, PTN, NTN1, NTN3, and SMOC1. Moreover, many of the enriched proteins in AD were validated in the detergent-insoluble proteome by five steps of differential extraction, proteomic analysis, or immunoblotting. Our study reveals a resource list of proteins and pathways that are exclusively present in the detergent-insoluble proteome, providing novel molecular insights to the formation of protein pathology in AD.

The U1 antisense morpholino oligonucleotide (AMO) disrupts U1 snRNP structure to promote intronic PCPA modification of pre-mRNAs.

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 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 interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 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 chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.

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

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.

An In Vivo CRISPR Screen Identifies That SNRPC Promotes Triple-Negative Breast Cancer Progression.

Dysregulation of RNA-binding proteins (RBP) is one of the characteristics of cancer. Investigating the biological functions and molecular mechanisms of abnormal RBPs can help uncover new cancer biomarkers and treatment strategies. To identify oncogenic RBPs in triple-negative breast cancer (TNBC), we employed an in vivo CRISPR screen and a TNBC progression model, which revealed small nuclear ribonucleoprotein polypeptide C (SNRPC), a subunit of the U1 small nuclear ribonucleoprotein particle (U1 snRNP), as a key modulator of TNBC progression. SNRPC was frequently upregulated, which corresponded to poor prognosis in patients with TNBC. SNRPC ablation significantly impaired the proliferation, migration, and invasion of TNBC cells in vitro and in vivo. In addition, SNRPC was essential for the stability of U1 snRNP and contributed to the RNA Pol II-controlled transcriptional program. Knockdown of SNRPC decreased RNA Pol II enrichment on a subset of oncogenes (TNFAIP2, E2F2, and CDK4) and reduced their expression levels. Furthermore, SNRPC deletion was confirmed to inhibit TNBC progression partially through regulation of the TNFAIP2-Rac1-ß-catenin signaling pathway. Taken together, this data suggests that SNRPC plays an oncogenic role in TNBC, is a marker of poor prognosis, and may be a valuable therapeutic target for patients with intractable TNBC. SIGNIFICANCE: A functional CRISPR screen identifies SNRPC as an RNA-binding protein that promotes the aggressiveness of breast cancer by facilitating Pol II-controlled transcription of oncogenes.

U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes.

The expansion of introns within mammalian genomes poses a challenge for the production of full-length messenger RNAs (mRNAs), with increasing evidence that these long AT-rich sequences present obstacles to transcription. Here, we investigate RNA polymerase II (RNAPII) elongation at high resolution in mammalian cells and demonstrate that RNAPII transcribes faster across introns. Moreover, we find that this acceleration requires the association of U1 snRNP (U1) with the elongation complex at 5' splice sites. The role of U1 to stimulate elongation rate through introns reduces the frequency of both premature termination and transcriptional arrest, thereby dramatically increasing RNA production. We further show that changes in RNAPII elongation rate due to AT content and U1 binding explain previous reports of pausing or termination at splice junctions and the edge of CpG islands. We propose that U1-mediated acceleration of elongation has evolved to mitigate the risks that long AT-rich introns pose to transcript completion.

Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.