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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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-β-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.
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.

Recent proteome and transcriptome profiling of Alzheimer\'s disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, erroneous splicing events, neuronal degeneration and cognitive impairment. Specifically, N40K-Tg shows the reduction of GABAergic synapse components (e.g., the GABA receptor subunit of GABRA2), and concomitant postsynaptic hyperexcitability that is rescued by a GABA receptor agonist. Crossing of N40K-Tg and the 5xFAD amyloidosis model indicates that the RNA splicing defect synergizes with the amyloid cascade to remodel the brain transcriptome and proteome, deregulate synaptic proteins, and accelerate cognitive decline. Thus, our results support the contribution of U1 snRNP-mediated splicing dysfunction to AD pathogenesis.

In eukaryotic cells, both alternative splicing and alternative polyadenylation (APA) play essential roles in the gene regulation network. U1 small ribonucleoprotein particle (U1 snRNP) is a major component of spliceosome, and U1 snRNP complex can suppress proximal APA sites through crosstalking with 3\' end processing factors. However, here we show that both knockdown and overexpression of SNRPA, SNRPC, SNRNP70, and SNRPD2, the U1 snRNP proteins, promote the usage of proximal APA sites at the transcriptome level. SNRNP70 can drive the phase transition of PABPN1 from droplet to aggregate, which may reduce the repressive effects of PABPN1 on the proximal APA sites. Additionally, SNRNP70 can also promote the proximal APA sites by recruiting CPSF6, suggesting that the function of CPSF6 on APA is related with other RNA-binding proteins and cell context-dependent. Consequently, these results reveal that, on the contrary to U1 snRNP complex, the free proteins of U1 snRNP complex can promote proximal APA sites through the interaction with 3\' end processing machinery.

Circular RNA (circRNA) has been proved to be a key regulator of gastric cancer (GC) progression. Circ_0009910 was found to be highly expressed in GC and related to GC progression, but its role and mechanism in GC progression need to be further improved. Our study aims to further reveal circ_0009910 roles in GC progression and elucidate its potential molecular mechanism. The expression of circ_0009910, microRNA (miR)-361-3p, and small nuclear ribonucleoprotein polypeptide A (SNRPA) mRNA was measured by quantitative real-time PCR. Protein expression was determined using western blot analysis. Cell proliferation, migration, invasion, and apoptosis were evaluated using EDU staining, transwell assay, and flow cytometry. Cell glycolysis were assessed by detecting glucose consumption, lactate production, and glycolysis-related markers protein expression. The relationship between miR-361-3p and circ_0009910 or SNRPA was confirmed by RNA pull-down assay and dual-luciferase reporter assay. In vivo experiments were performed to explore the effect of circ_0009910 silencing on GC tumorigenesis. Circ_0009910 and SNRPA were upregulated in GC tumor tissues and cells. Knockdown of circ_0009910 or SNRPA could inhibit GC cell proliferation, migration, invasion, glycolysis, and promote apoptosis. Circ_0009910 could sponge miR-361-3p, and miR-361-3p could target SNRPA. Further experiments confirmed that circ_0009910 positively regulated SNRPA by sponging miR-361-3p. Additionally, SNRPA overexpression abolished the negative regulation of circ_0009910 silencing on GC progression. Furthermore, silenced circ_0009910 also reduced GC tumorigenesis in vivo. Our data showed that circ_0009910 might be a target for GC treatment, which could promote GC proliferation, metastasis, and glycolysis by the miR-361-3p/SNRPA axis.

Eukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.

Riboswitches are important gene regulatory elements frequently encountered in bacterial mRNAs. The recently discovered nadA riboswitch contains two similar, tandemly arrayed aptamer domains, with the first domain possessing high affinity for nicotinamide adenine dinucleotide (NAD+). The second domain which comprises the ribosomal binding site in a putative regulatory helix, however, has withdrawn from detection of ligand-induced structural modulation thus far, and therefore, the identity of the cognate ligand and the regulation mechanism have remained unclear. Here, we report crystal structures of both riboswitch domains, each bound to NAD+. Furthermore, we demonstrate that ligand binding to domain 2 requires significantly higher concentrations of NAD+ (or ADP retaining analogs) compared to domain 1. Using a fluorescence spectroscopic approach, we further shed light on the structural features which are responsible for the different ligand affinities, and describe the Mg2+-dependent, distinct folding and pre-organization of their binding pockets. Finally, we speculate about possible scenarios for nadA RNA gene regulation as a putative two-concentration sensor module for a time-controlled signal that is primed and stalled by the gene regulation machinery at low ligand concentrations (domain 1), and finally triggers repression of translation as soon as high ligand concentrations are reached in the cell (domain 2).