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Molecular Cell Aug 2023Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice...
Splicing of pre-mRNAs critically contributes to gene regulation and proteome expansion in eukaryotes, but our understanding of the recognition and pairing of splice sites during spliceosome assembly lacks detail. Here, we identify the multidomain RNA-binding protein FUBP1 as a key splicing factor that binds to a hitherto unknown cis-regulatory motif. By collecting NMR, structural, and in vivo interaction data, we demonstrate that FUBP1 stabilizes U2AF2 and SF1, key components at the 3' splice site, through multivalent binding interfaces located within its disordered regions. Transcriptional profiling and kinetic modeling reveal that FUBP1 is required for efficient splicing of long introns, which is impaired in cancer patients harboring FUBP1 mutations. Notably, FUBP1 interacts with numerous U1 snRNP-associated proteins, suggesting a unique role for FUBP1 in splice site bridging for long introns. We propose a compelling model for 3' splice site recognition of long introns, which represent 80% of all human introns.
Topics: Humans; RNA Splice Sites; Introns; RNA Splicing Factors; RNA Splicing; RNA-Binding Proteins; RNA Precursors; DNA-Binding Proteins
PubMed: 37506698
DOI: 10.1016/j.molcel.2023.07.002 -
Molecular Cell Apr 2023RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working...
RNA-binding proteins (RBPs) bind at different positions of the pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These "positional rules" can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease.
Topics: Humans; RNA; HeLa Cells; Polypyrimidine Tract-Binding Protein; RNA Splicing; RNA-Binding Proteins; RNA Precursors; Alternative Splicing; Heterogeneous-Nuclear Ribonucleoproteins; Serine-Arginine Splicing Factors
PubMed: 36958328
DOI: 10.1016/j.molcel.2023.03.001 -
Cell Research Mar 2022In response to DNA double-strand breaks (DSBs), DNA damage repair factors are recruited to DNA lesions and form nuclear foci. However, the underlying molecular mechanism...
In response to DNA double-strand breaks (DSBs), DNA damage repair factors are recruited to DNA lesions and form nuclear foci. However, the underlying molecular mechanism remains largely elusive. Here, by analyzing the localization of DSB repair factors in the XY body and DSB foci, we demonstrate that pre-ribosomal RNA (pre-rRNA) mediates the recruitment of DSB repair factors around DNA lesions. Pre-rRNA exists in the XY body, a DSB repair hub, during meiotic prophase, and colocalizes with DSB repair factors, such as MDC1, BRCA1 and TopBP1. Moreover, pre-rRNA-associated proteins and RNAs, such as ribosomal protein subunits, RNase MRP and snoRNAs, also localize in the XY body. Similar to those in the XY body, pre-rRNA and ribosomal proteins also localize at DSB foci and associate with DSB repair factors. RNA polymerase I inhibitor treatment that transiently suppresses transcription of rDNA but does not affect global protein translation abolishes foci formation of DSB repair factors as well as DSB repair. The FHA domain and PST repeats of MDC1 recognize pre-rRNA and mediate phase separation of DSB repair factors, which may be the molecular basis for the foci formation of DSB repair factors during DSB response.
Topics: Cell Cycle Proteins; DNA; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Meiosis; Prophase; RNA Precursors; RNA, Ribosomal
PubMed: 34980897
DOI: 10.1038/s41422-021-00597-4 -
Molecular Cell Dec 2019Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and...
Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.
Topics: Active Transport, Cell Nucleus; Antigens, Nuclear; Cell Nucleolus; Chromosomal Proteins, Non-Histone; Female; HEK293 Cells; HeLa Cells; Humans; Nucleic Acid Conformation; Protein Binding; Protein Interaction Domains and Motifs; RNA Precursors; RNA Processing, Post-Transcriptional; RNA, Ribosomal
PubMed: 31540874
DOI: 10.1016/j.molcel.2019.08.014 -
The FEBS Journal Jul 2022Coordination of transcription and processing of RNA is a basic principle in regulation of gene expression in eukaryotes. In the case of mRNA, coordination is primarily... (Review)
Review
Coordination of transcription and processing of RNA is a basic principle in regulation of gene expression in eukaryotes. In the case of mRNA, coordination is primarily founded on a co-transcriptional processing mechanism by which a nascent precursor mRNA undergoes maturation via cleavage and modification by the transcription machinery. A similar mechanism controls the biosynthesis of rRNA. However, the coordination of transcription and processing of tRNA, a rather short transcript, remains unknown. Here, we present a model for high molecular weight initiation complexes of human RNA polymerase III that assemble on tRNA genes and process precursor transcripts to mature forms. These multifunctional initiation complexes may support co-transcriptional processing, such as the removal of the 5' leader of precursor tRNA by RNase P. Based on this model, maturation of tRNA is predetermined prior to transcription initiation.
Topics: Humans; RNA Polymerase III; RNA Precursors; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Transfer; Ribonuclease P; Transcription, Genetic
PubMed: 33929081
DOI: 10.1111/febs.15904 -
Current Opinion in Plant Biology Oct 2021Light signal perceived by the red/far-red absorbing phytochrome (phy) family of photoreceptors regulates plant growth and development throughout the life cycle.... (Review)
Review
Light signal perceived by the red/far-red absorbing phytochrome (phy) family of photoreceptors regulates plant growth and development throughout the life cycle. Phytochromes regulate the light-triggered physiological responses by controlling gene expression both at the transcriptional and post-transcriptional levels. Recent large-scale RNA-seq studies have demonstrated the roles of phys in altering the global transcript diversity by modulating the pre-mRNA splicing in response to light. Moreover, several phy-interacting splicing factors/regulators from different species have been identified using forward genetics and protein-protein interaction studies, which modulate the light-regulated pre-mRNA splicing. In this article, we summarize our current understanding of the role of phys in the light-mediated pre-mRNA splicing and how that contributes to the regulation of gene expression to promote photomorphogenesis.
Topics: Arabidopsis; Arabidopsis Proteins; Light; Phytochrome; RNA Precursors; RNA Splicing
PubMed: 33823333
DOI: 10.1016/j.pbi.2021.102037 -
Genes & Development Dec 2023RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA... (Review)
Review
RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA complexes remains poorly understood. In this issue of , Zhang and colleagues (pp. 968-983) combine biochemical experiments with AI-based structure prediction methods to generate a model for the interaction between SF3B1, a core splicing factor essential for the recognition of the intron branchpoint, and SUGP1, a protein that bridges SF3B1 with the helicase DHX15. Interaction with SF3B1 exposes the G-patch domain of SUGP1, facilitating binding to and activation of DHX15. The model can explain the activation of cryptic 3' splice sites induced by mutations in SF3B1 or SUGP1 frequently found in cancer.
Topics: RNA Splicing; Spliceosomes; RNA Splicing Factors; RNA Splice Sites; RNA Precursors; Artificial Intelligence; Mutation; Phosphoproteins
PubMed: 38092520
DOI: 10.1101/gad.351373.123 -
International Journal of Molecular... Sep 2022Poly(A) tails are present on almost all eukaryotic mRNAs, and play critical roles in mRNA stability, nuclear export, and translation efficiency. The biosynthesis and... (Review)
Review
Poly(A) tails are present on almost all eukaryotic mRNAs, and play critical roles in mRNA stability, nuclear export, and translation efficiency. The biosynthesis and shortening of a poly(A) tail are regulated by large multiprotein complexes. However, the molecular mechanisms of these protein machineries still remain unclear. Recent studies regarding the structural and biochemical characteristics of those protein complexes have shed light on the potential mechanisms of polyadenylation and deadenylation. This review summarizes the recent structural studies on pre-mRNA 3'-end processing complexes that initiate the polyadenylation and discusses the similarities and differences between yeast and human machineries. Specifically, we highlight recent biochemical efforts in the reconstitution of the active human canonical pre-mRNA 3'-end processing systems, as well as the roles of RBBP6/Mpe1 in activating the entire machinery. We also describe how poly(A) tails are removed by the PAN2-PAN3 and CCR4-NOT deadenylation complexes and discuss the emerging role of the cytoplasmic poly(A)-binding protein (PABPC) in promoting deadenylation. Together, these recent discoveries show that the dynamic features of these machineries play important roles in regulating polyadenylation and deadenylation.
Topics: DNA-Binding Proteins; Exoribonucleases; Humans; Multiprotein Complexes; Poly(A)-Binding Proteins; Polyadenylation; RNA Precursors; RNA Stability; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases; mRNA Cleavage and Polyadenylation Factors
PubMed: 36232288
DOI: 10.3390/ijms231910985 -
Nature Communications Feb 2023Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this...
Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5'end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3'end processing performed by RBM7. Altogether, our data provide a temporal view of RNA-protein interactions during the early phases of transcription.
Topics: RNA-Binding Proteins; Cell Nucleus; RNA Precursors; RNA Splicing; RNA Polymerase II; RNA, Small Nucleolar
PubMed: 36755023
DOI: 10.1038/s41467-023-36345-y -
Molecular Cell Apr 2023Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease...
Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease (TSEN) comprising four subunits: TSEN2, TSEN15, TSEN34, and TSEN54. Here, we report the cryo-EM structures of human TSEN bound to full-length pre-tRNA in the pre-catalytic and post-catalytic states at average resolutions of 2.94 and 2.88 Å, respectively. Human TSEN features an extended surface groove that holds the L-shaped pre-tRNA. The mature domain of pre-tRNA is recognized by conserved structural elements of TSEN34, TSEN54, and TSEN2. Such recognition orients the anticodon stem of pre-tRNA and places the 3'-splice site and 5'-splice site into the catalytic centers of TSEN34 and TSEN2, respectively. The bulk of the intron sequences makes no direct interaction with TSEN, explaining why pre-tRNAs of varying introns can be accommodated and cleaved. Our structures reveal the molecular ruler mechanism of pre-tRNA cleavage by TSEN.
Topics: Humans; Introns; RNA Precursors; Endoribonucleases; RNA, Transfer; RNA Splice Sites; RNA Splicing; Nucleic Acid Conformation; Endonucleases
PubMed: 37028420
DOI: 10.1016/j.molcel.2023.03.015