ABSTRACT
Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.
Subject(s)
Liver Failure, Acute , RNA Precursors , RNA Splicing Factors , RNA Splicing , RNA-Binding Proteins , RNA Splicing Factors/metabolism , RNA Splicing Factors/genetics , Animals , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA Splicing/genetics , Liver Failure, Acute/metabolism , Liver Failure, Acute/genetics , RNA Precursors/metabolism , RNA Precursors/genetics , Mice , Humans , Mice, Inbred C57BL , Male , Phosphoproteins/metabolism , Phosphoproteins/genetics , Disease Models, Animal , Protein Binding , Trans-ActivatorsABSTRACT
The eukaryotic mRNA surveillance pathway, a pivotal guardian of mRNA fidelity, stands at the nexus of diverse biological processes, including antiviral immunity. Despite the recognized function of splicing factors on mRNA fate, the intricate interplay shaping the mRNA surveillance pathway remains elusive. We illustrate that the conserved splicing factor U2 snRNP auxiliary factor large subunit B (U2AF65B) modulates splicing of mRNA surveillance complex, contributing to transcriptomic homeostasis in maize. The functionality of the mRNA surveillance pathway requires ZmU2AF65B-mediated normal splicing of upstream frameshift 3 (ZmUPF3) pre-mRNA, encoding a core factor in this pathway. Intriguingly, sugarcane mosaic virus (SCMV)-coded nuclear inclusion protein a protease (NIa-Pro) hinders the splicing function of ZmU2AF65B. Furthermore, NIa-Pro disrupts ZmU2AF65B binding to ZmUPF3 pre-mRNA, leading to dysregulated splicing of ZmUPF3 transcripts and, consequently, impairing mRNA surveillance, thus facilitating viral infection. Together, this study establishes that splicing governs the mRNA surveillance pathway and identifies a pathogenic protein capable of disrupting this regulation to compromise RNA immunity.
Subject(s)
Potyvirus , RNA Splicing , RNA, Messenger , Zea mays , Zea mays/virology , Zea mays/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Potyvirus/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Plant Diseases/virology , Plant Diseases/genetics , Splicing Factor U2AF/metabolism , Splicing Factor U2AF/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , Gene Expression Regulation, PlantABSTRACT
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.
Subject(s)
Ribonucleoprotein, U1 Small Nuclear , Serine-Arginine Splicing Factors , Spliceosomes , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/chemistry , Serine-Arginine Splicing Factors/genetics , Phosphorylation , Spliceosomes/metabolism , Spliceosomes/chemistry , Humans , Ribonucleoprotein, U1 Small Nuclear/metabolism , Ribonucleoprotein, U1 Small Nuclear/chemistry , Ribonucleoprotein, U1 Small Nuclear/genetics , RNA Splicing , Protein Binding , RNA Precursors/metabolism , RNA Precursors/genetics , RNA Precursors/chemistryABSTRACT
The twenty-three Fanconi anemia (FA) proteins cooperate in the FA/BRCA pathway to repair DNA interstrand cross-links (ICLs). The cell division cycle and apoptosis regulator 1 (CCAR1) protein is also a regulator of ICL repair, though its possible function in the FA/BRCA pathway remains unknown. Here, we demonstrate that CCAR1 plays a unique upstream role in the FA/BRCA pathway and is required for FANCA protein expression in human cells. Interestingly, CCAR1 co-immunoprecipitates with FANCA pre-mRNA and is required for FANCA mRNA processing. Loss of CCAR1 results in retention of a poison exon in the FANCA transcript, thereby leading to reduced FANCA protein expression. A unique domain of CCAR1, the EF hand domain, is required for interaction with the U2AF heterodimer of the spliceosome and for excision of the poison exon. Taken together, CCAR1 is a splicing modulator required for normal splicing of the FANCA mRNA and other mRNAs involved in various cellular pathways.
Subject(s)
Apoptosis Regulatory Proteins , Cell Cycle Proteins , Fanconi Anemia Complementation Group A Protein , Fanconi Anemia , RNA Splicing , Splicing Factor U2AF , Humans , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , BRCA2 Protein/metabolism , BRCA2 Protein/genetics , DNA Repair , Endodeoxyribonucleases , Exons , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia Complementation Group A Protein/metabolism , HEK293 Cells , HeLa Cells , Protein Binding , RNA Precursors/metabolism , RNA Precursors/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Spliceosomes/metabolism , Spliceosomes/genetics , Splicing Factor U2AF/metabolism , Splicing Factor U2AF/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolismABSTRACT
Alternative polyadenylation (APA) is a crucial mechanism for regulating gene expression during pre-mRNA 3' processing. Pre-mRNA 3' end processing factors is the main factor involved in this process. However, pre-mRNA 3' end processing factors in different cancer expression profiles and the relationship between pre-mRNA 3' end processing factors and tumor microenvironment and the prognosis of the same patient is still unclear. In this study, we conducted a comprehensive exploration of the core pre-mRNA 3' end processing factors across various cancer types by utilizing common cancer database, and revealing a robust correlation between the expression of these core factors and tumor characteristics. Leveraging advanced bioinformatics databases, we evaluated the expression levels and prognostic relevance of pre-mRNA 3' end processing factors across pan-cancer tissues. Our extensive pan-cancer analysis revealed unique expression patterns of pre-mRNA 3' end processing factors in both tumor and adjacent non-tumorous tissues. Notably, we found a significant correlation between the expression levels of pre-mRNA 3' end processing factors and patient prognosis. Furthermore, we identified strong associations between pre-mRNA 3' end processing factors expression and various factors, such as stromal, immune, RNA stemness, and DNA stemness scores across pan-cancer tissues. Our data also highlighted a link between the expression of pre-mRNA 3' end processing factors and sensitivity to specific drugs, including pyrazoloacndine, amonaflide, and chelerythrinede, among others. We found four key pre-mRNA 3' end processing factors that play a crucial role in mRNA preprocessing. Our study illuminates the potential promotion and inhibition role of pre-mRNA 3' end processing regulators in the progression of cancer, CPSF2, CPSF3, CSTF2, SYMPK offering valuable insights for future research investigations on these regulators as diagnostic markers and therapeutic targets across pan-cancer.
Subject(s)
Neoplasms , RNA Precursors , Tumor Microenvironment , Humans , Tumor Microenvironment/genetics , Neoplasms/genetics , Neoplasms/pathology , Prognosis , RNA Precursors/genetics , RNA Precursors/metabolism , Gene Expression Regulation, Neoplastic , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA 3' End Processing/genetics , Computational Biology/methods , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , PolyadenylationABSTRACT
The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.
Subject(s)
Cell Nucleus , RNA Splicing , RNA-Binding Proteins , Humans , Cell Nucleus/metabolism , Cell Nucleus/genetics , Animals , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Mice , RNA Precursors/metabolism , RNA Precursors/genetics , Mutation , Spliceosomes/metabolism , Spliceosomes/genetics , HeLa Cells , HEK293 CellsABSTRACT
The Drosophila RNA-binding protein (RBP) Nab2 acts in neurons to regulate neurodevelopment and is orthologous to the human intellectual disability-linked RBP, ZC3H14. Nab2 governs axon projection in mushroom body neurons and limits dendritic arborization of class IV sensory neurons in part by regulating splicing events in â¼150 mRNAs. Analysis of the Sex-lethal (Sxl) mRNA revealed that Nab2 promotes an exon-skipping event and regulates m6A methylation on Sxl pre-mRNA by the Mettl3 methyltransferase. Mettl3 heterozygosity broadly rescues Nab2null phenotypes implying that Nab2 acts through similar mechanisms on other RNAs, including unidentified targets involved in neurodevelopment. Here, we show that Nab2 and Mettl3 regulate the removal of a 5'UTR (untranslated region) intron in the trio pre-mRNA. Trio utilizes two GEF domains to balance Rac and RhoGTPase activity. Intriguingly, an isoform of Trio containing only the RhoGEF domain, GEF2, is depleted in Nab2null nervous tissue. Expression of Trio-GEF2 rescues projection defects in Nab2null axons and dendrites, while the GEF1 Rac1-regulatory domain exacerbates these defects, suggesting Nab2-mediated regulation Trio-GEF activities. Collectively, these data indicate that Nab2-regulated processing of trio is critical for balancing Trio-GEF1 and -GEF2 activity and show that Nab2, Mettl3, and Trio function in a common pathway that shapes axon and dendrite morphology.
Subject(s)
Axons , Dendrites , Drosophila Proteins , Drosophila melanogaster , Guanine Nucleotide Exchange Factors , RNA-Binding Proteins , Animals , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Axons/metabolism , Dendrites/metabolism , Drosophila melanogaster/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Guanine Nucleotide Exchange Factors/genetics , Methyltransferases/metabolism , Methyltransferases/genetics , RNA Splicing , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA Precursors/metabolism , RNA Precursors/geneticsABSTRACT
The spliceosome executes pre-mRNA splicing through four sequential stages: assembly, activation, catalysis, and disassembly. Activation of the spliceosome, namely remodeling of the pre-catalytic spliceosome (B complex) into the activated spliceosome (Bact complex) and the catalytically activated spliceosome (B* complex), involves major flux of protein components and structural rearrangements. Relying on a splicing inhibitor, we have captured six intermediate states between the B and B* complexes: pre-Bact, Bact-I, Bact-II, Bact-III, Bact-IV, and post-Bact. Their cryo-EM structures, together with an improved structure of the catalytic step I spliceosome (C complex), reveal how the catalytic center matures around the internal stem loop of U6 snRNA, how the branch site approaches 5'-splice site, how the RNA helicase PRP2 rearranges to bind pre-mRNA, and how U2 snRNP undergoes remarkable movement to facilitate activation. We identify a previously unrecognized key role of PRP2 in spliceosome activation. Our study recapitulates a molecular choreography of the human spliceosome during its catalytic activation.
Subject(s)
Cryoelectron Microscopy , RNA Precursors , RNA Splicing , RNA, Small Nuclear , Spliceosomes , Spliceosomes/metabolism , Humans , RNA Precursors/metabolism , RNA Precursors/genetics , RNA, Small Nuclear/metabolism , Ribonucleoprotein, U2 Small Nuclear/metabolism , Ribonucleoprotein, U2 Small Nuclear/genetics , Models, Molecular , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Catalytic DomainABSTRACT
Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.
Subject(s)
Alternative Splicing , MicroRNAs , Ribonuclease III , MicroRNAs/genetics , Humans , Ribonuclease III/genetics , Ribonuclease III/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splice Sites/genetics , Mutation , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Introns/geneticsABSTRACT
Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.
Subject(s)
RNA Precursors , RNA Splicing , RNA, Small Nuclear , RNA, Small Nuclear/genetics , Animals , Mice , Humans , RNA Precursors/genetics , RNA Precursors/metabolism , Base Sequence , Exons/genetics , RNA, Antisense/geneticsABSTRACT
MicroRNAs (miRNAs) are generated from stem-loop-structured double-stranded RNA precursors by the consecutive action of the two RNase III-type endoribonuclease Drosha and Dicer. However, such structures are very common on cellular transcripts and specific features have evolved that guide and regulate processing of stem-loop-structured hairpins into mature and functional miRNAs. These features include sequence motifs and local RNA structures but also trans-acting factors such as RNA binding proteins. The menu of features required for miRNA biogenesis is summarized in this review.
Subject(s)
MicroRNAs , Ribonuclease III , MicroRNAs/metabolism , MicroRNAs/genetics , Humans , Animals , Ribonuclease III/metabolism , Ribonuclease III/genetics , RNA, Double-Stranded/metabolism , RNA, Double-Stranded/genetics , RNA Precursors/metabolism , RNA Precursors/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Nucleic Acid ConformationABSTRACT
BACKGROUND: The progression of non-small cell lung cancer (NSCLC) is significantly influenced by circular RNAs (circRNAs), especially in tumor hypoxia microenvironment. However, the precise functions and underlying mechanisms of dysregulated circRNAs in NSCLC remain largely unexplored. METHODS: Differentially expressed circRNAs in NSCLC tissues were identified through high-throughput RNA sequencing. The characteristics of circ_0007386 were rigorously confirmed via Sanger sequencing, RNase R treatment and actinomycin D treatment. The effects of circ_0007386 on proliferation and apoptosis were investigated using CCK8, cloning formation assays, TUNEL staining, and flow cytometry assays in vitro. In vivo, xenograft tumor models were used to evaluate its impact on proliferation. Mechanistically, the regulatory relationships of circ_0007386, miR-383-5p and CIRBP were examined through dual luciferase reporter assays and rescue experiments. Additionally, we detected the binding of EIF4A3 to CRIM1 pre-mRNA by RNA immunoprecipitation and the interaction between YAP1 and EIF4A3 under hypoxic conditions by co-immunoprecipitation. RESULTS: Our investigation revealed a novel circRNA, designated as circ_0007386, that was upregulated in NSCLC tissues and cell lines. Circ_0007386 modulated proliferation and apoptosis in NSCLC both in vitro and in vivo. Functionally, circ_0007386 acted as a sponge for miR-383-5p, targeting CIRBP, which influenced NSCLC cell proliferation and apoptosis via the PI3K/AKT signaling pathway. Furthermore, under hypoxic conditions, the interaction between YAP1 and EIF4A3 was enhanced, leading to the displacement of EIF4A4 from binding to CRIM1 pre-mRNA. This facilitated the back-splicing of CRIM1 pre-mRNA, increasing the formation of circ_0007386. The circ_0007386/miR-383-5p/CIRBP axis was significantly associated with the clinical features and prognosis of NSCLC patients. CONCLUSIONS: Circ_0007386, regulated by YAP1-EIF4A3 interaction under hypoxia conditions, plays an oncogenic role in NSCLC progression via the miR-383-5p/CIRBP axis.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Disease Progression , Eukaryotic Initiation Factor-4A , Lung Neoplasms , RNA, Circular , YAP-Signaling Proteins , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , RNA, Circular/genetics , RNA, Circular/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Animals , YAP-Signaling Proteins/metabolism , Mice , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4A/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Female , Cell Line, Tumor , Cell Proliferation , RNA Precursors/metabolism , RNA Precursors/genetics , Male , RNA Splicing , Apoptosis , MicroRNAs/genetics , MicroRNAs/metabolism , Mice, Nude , Gene Expression Regulation, Neoplastic , DEAD-box RNA HelicasesABSTRACT
Pre-mRNA splicing, the removal of introns and ligation of flanking exons, is a crucial step in eukaryotic gene expression. The spliceosome, a macromolecular complex made up of five small nuclear RNAs (snRNAs) and dozens of proteins, assembles on introns via a complex pathway before catalyzing the two transesterification reactions necessary for splicing. All of these steps have the potential to be highly regulated to ensure correct mRNA isoform production for proper cellular function. While Saccharomyces cerevisiae (yeast) has a limited set of intron-containing genes, many of these genes are highly expressed, resulting in a large number of transcripts in a cell being spliced. As a result, splicing regulation is of critical importance for yeast. Just as in humans, yeast splicing can be influenced by protein components of the splicing machinery, structures and properties of the pre-mRNA itself, or by the action of trans-acting factors. It is likely that further analysis of the mechanisms and pathways of splicing regulation in yeast can reveal general principles applicable to other eukaryotes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.
Subject(s)
RNA Precursors , RNA Splicing , Saccharomyces cerevisiae , Spliceosomes , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spliceosomes/metabolism , Spliceosomes/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Pre-mRNA splicing is a significant step for post-transcriptional modifications and functions in a wide range of physiological processes in plants. Human NHP2L binds to U4 snRNA during spliceosome assembly; it is involved in RNA splicing and mediates the development of human tumors. However, no ortholog has yet been identified in plants. Therefore, we report At4g12600 encoding the ortholog NHP2L protein, and AtSNU13 associates with the component of the spliceosome complex; the atsnu13 mutant showed compromised resistance in disease resistance, indicating that AtSNU13 is a positive regulator of plant immunity. Compared to wild-type plants, the atsnu13 mutation resulted in altered splicing patterns for defense-related genes and decreased expression of defense-related genes, such as RBOHD and ALD1. Further investigation shows that AtSNU13 promotes the interaction between U4/U6.U5 tri-snRNP-specific 27 K and the motif in target mRNAs to regulate the RNA splicing. Our study highlights the role of AtSNU13 in regulating plant immunity by affecting the pre-mRNA splicing of defense-related genes.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Plant Immunity , RNA Precursors , RNA Splicing , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Plant Diseases/genetics , Plant Diseases/immunology , Plant Immunity/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , Spliceosomes/metabolism , Spliceosomes/geneticsABSTRACT
The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.
Subject(s)
Mitochondria , RNA, Transfer , Ribonuclease P , tRNA Methyltransferases , Humans , RNA, Transfer/metabolism , RNA, Transfer/genetics , RNA, Transfer/chemistry , Mitochondria/metabolism , Ribonuclease P/metabolism , Ribonuclease P/genetics , Ribonuclease P/chemistry , tRNA Methyltransferases/metabolism , tRNA Methyltransferases/genetics , tRNA Methyltransferases/chemistry , RNA Processing, Post-Transcriptional , Cryoelectron Microscopy , RNA, Mitochondrial/metabolism , RNA, Mitochondrial/genetics , RNA, Mitochondrial/chemistry , Methylation , Nucleic Acid Conformation , Models, Molecular , RNA Precursors/metabolism , RNA Precursors/geneticsABSTRACT
MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases, including cancers. In our investigation focusing on altering specific miRNA expression in human pancreatic cancer cells, we encountered an interesting finding. While two expression vector designs effectively enhanced miR-708 levels, they were unable to elevate mature forms of miR-29b, -1290, -2467, and -6831 in pancreatic cancer cell lines. This finding was also observed in a panel of other non-pancreatic cancer cell lines, suggesting that miRNA processing efficiency was cell line specific. Using a step-by-step approach in each step of miRNA processing, we ruled out alternative strand selection by the RISC complex and transcriptional interference at the primary miRNA (pri-miRNA) level. DROSHA processing and pri-miRNA export from the nucleus also appeared to be occurring normally. We observed precursor (pre-miRNA) accumulation only in cell lines where mature miRNA expression was not achieved, suggesting that the block was occurring at the pre-miRNA stage. To further confirm this, synthetic pre-miRNA mimics that bypass DICER processing were processed into mature miRNAs in all cases. This study has demonstrated the distinct behaviours of different miRNAs with the same vector in the same cell line, the same miRNA between the two vector designs, and with the same miRNA across different cell lines. We identified a stable vector pre-miRNA processing block. Our findings on the structural and sequence differences between successful and non-successful vector designs could help to inform future chimeric miRNA design strategies and act as a guide to other researchers on the intricate processing dynamics that can impact vector efficiency. Our research confirms the potential of miRNA mimics to surmount some of these complexities.
Subject(s)
MicroRNAs , Pancreatic Neoplasms , RNA Processing, Post-Transcriptional , MicroRNAs/genetics , MicroRNAs/metabolism , Humans , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , RNA Processing, Post-Transcriptional/genetics , Cell Line, Tumor , Ribonuclease III/metabolism , Ribonuclease III/genetics , Gene Expression Regulation, Neoplastic , Transfection , RNA Precursors/genetics , RNA Precursors/metabolism , AnimalsABSTRACT
Precursor-mRNA (pre-mRNA) splicing requires the assembly, remodelling and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome1. Recent studies have shed light on spliceosome assembly and remodelling for catalysis2-6, but the mechanism of disassembly remains unclear. Here we report cryo-electron microscopy structures of nematode and human terminal intron lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 therefore controls both the start5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of the initiation of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes.
Subject(s)
Caenorhabditis elegans , Spliceosomes , Animals , Humans , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cryoelectron Microscopy , Introns/genetics , Models, Molecular , RNA Helicases/metabolism , RNA Precursors/metabolism , RNA Precursors/genetics , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Nuclear/metabolism , RNA, Small Nuclear/chemistry , Spliceosomes/metabolism , Spliceosomes/ultrastructure , Spliceosomes/chemistry , RNA Splicing Factors/metabolism , RNA-Binding Proteins/metabolismABSTRACT
N6-methyladenosine (m6A) is the most abundant modification to mRNAs. Loss-of-function studies of main m6A regulators have indicated the role of m6A in pre-mRNA splicing. Recent studies have reported the role of splicing in preventing m6A deposition. Understanding the interplay between m6A and mRNA splicing holds the potential to clarify the significance of these fundamental molecular mechanisms in cell development and function, thereby shedding light on their involvement in the pathogenesis of myriad diseases.
Subject(s)
Adenosine , RNA Methylation , RNA Splicing , RNA, Messenger , Animals , Humans , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The essential role of U4 snRNP in pre-messenger RNA (mRNA) splicing has been well established. In this study, we utilized an antisense morpholino oligonucleotide (AMO) specifically targeting U4 snRNA to achieve functional knockdown of U4 snRNP in HeLa cells. Our results showed that this knockdown resulted in global intronic premature cleavage and polyadenylation (PCPA) events, comparable to the effects observed with U1 AMO treatment, as demonstrated by mRNA 3'-seq analysis. Furthermore, our study suggested that this may be a common phenomenon in both human and mouse cell lines. Additionally, we showed that U4 AMO treatment disrupted transcription elongation, as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis for RNAPII. Collectively, our results identified a unique role for U4 snRNP in the inhibition of PCPA and indicated a model wherein splicing intrinsically inhibits intronic cleavage and polyadenylation in the context of cotranscriptional mRNA processing.
Subject(s)
Polyadenylation , RNA Precursors , RNA Splicing , Humans , RNA Precursors/metabolism , RNA Precursors/genetics , HeLa Cells , Mice , Animals , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Introns/geneticsABSTRACT
Ribonuclease P (RNase P) was first described in the 1970's as an endoribonuclease acting in the maturation of precursor transfer RNAs (tRNAs). More recent studies, however, have uncovered non-canonical roles for RNase P and its components. Here, we review the recent progress of its involvement in chromatin assembly, DNA damage response, and maintenance of genome stability with implications in tumorigenesis. The possibility of RNase P as a therapeutic target in cancer is also discussed.