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Cell Jun 2022Most circular RNAs are produced from the back-splicing of exons of precursor mRNAs. Recent technological advances have in part overcome problems with their circular... (Review)
Review
Most circular RNAs are produced from the back-splicing of exons of precursor mRNAs. Recent technological advances have in part overcome problems with their circular conformation and sequence overlap with linear cognate mRNAs, allowing a better understanding of their cellular roles. Depending on their localization and specific interactions with DNA, RNA, and proteins, circular RNAs can modulate transcription and splicing, regulate stability and translation of cytoplasmic mRNAs, interfere with signaling pathways, and serve as templates for translation in different biological and pathophysiological contexts. Emerging applications of RNA circles to interfere with cellular processes, modulate immune responses, and direct translation into proteins shed new light on biomedical research. In this review, we discuss approaches used in circular RNA studies and the current understanding of their regulatory roles and potential applications.
Topics: Proteins; RNA; RNA Precursors; RNA Splicing; RNA, Circular; RNA, Messenger
PubMed: 35584701
DOI: 10.1016/j.cell.2022.04.021 -
Science (New York, N.Y.) Mar 2023Loss of nuclear TDP-43 is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)....
Loss of nuclear TDP-43 is a hallmark of neurodegeneration in TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 mislocalization results in cryptic splicing and polyadenylation of pre-messenger RNAs (pre-mRNAs) encoding stathmin-2 (also known as SCG10), a protein that is required for axonal regeneration. We found that TDP-43 binding to a GU-rich region sterically blocked recognition of the cryptic 3' splice site in pre-mRNA. Targeting dCasRx or antisense oligonucleotides (ASOs) suppressed cryptic splicing, which restored axonal regeneration and stathmin-2-dependent lysosome trafficking in TDP-43-deficient human motor neurons. In mice that were gene-edited to contain human cryptic splice-polyadenylation sequences, ASO injection into cerebral spinal fluid successfully corrected pre-mRNA misprocessing and restored stathmin-2 expression levels independently of TDP-43 binding.
Topics: Animals; Humans; Mice; DNA-Binding Proteins; Polyadenylation; RNA Precursors; Stathmin; TDP-43 Proteinopathies; RNA Splicing; RNA Splice Sites; Gene Editing; Oligonucleotides, Antisense; Neuronal Outgrowth
PubMed: 36927019
DOI: 10.1126/science.abq5622 -
Annual Review of Biochemistry Jun 2023Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave... (Review)
Review
Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.
Topics: Humans; RNA, Messenger; RNA Precursors; mRNA Cleavage and Polyadenylation Factors; Saccharomyces cerevisiae; Gene Expression
PubMed: 37001138
DOI: 10.1146/annurev-biochem-052521-012445 -
Molecular Cell Feb 2022Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become...
Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish a vast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.
Topics: Alternative Splicing; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; HEK293 Cells; Hep G2 Cells; Humans; Intramolecular Transferases; Liver Neoplasms; RNA 3' End Processing; RNA Precursors; RNA, Messenger; Transcription, Genetic
PubMed: 35051350
DOI: 10.1016/j.molcel.2021.12.023 -
Genes Feb 2022Alternative splicing of pre-mRNA is a key mechanism for increasing the complexity of proteins in humans, causing a diversity of expression of transcriptomes and... (Review)
Review
Alternative splicing of pre-mRNA is a key mechanism for increasing the complexity of proteins in humans, causing a diversity of expression of transcriptomes and proteomes in a tissue-specific manner. Alternative splicing is regulated by a variety of splicing factors. However, the changes and errors of splicing regulation caused by splicing factors are strongly related to many diseases, something which represents one of this study's main interests. Further understanding of alternative splicing regulation mediated by cellular factors is also a prospective choice to develop specific drugs for targeting the dynamic RNA splicing process. In this review, we firstly concluded the basic principle of alternative splicing. Afterwards, we showed how splicing isoforms affect physiological activities through specific disease examples. Finally, the available treatment methods relative to adjusting splicing activities have been summarized.
Topics: Alternative Splicing; Humans; Prospective Studies; Protein Isoforms; RNA Precursors; RNA Splicing Factors
PubMed: 35327956
DOI: 10.3390/genes13030401 -
Molecular Cell Jan 2022Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize... (Comparative Study)
Comparative Study
Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 10- to 10-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.
Topics: A549 Cells; Bacteriophage T4; HEK293 Cells; HeLa Cells; Humans; Immunity, Innate; Introns; Nucleic Acid Conformation; Protein Kinase Inhibitors; RNA Ligase (ATP); RNA Precursors; RNA, Circular; Thymidylate Synthase; Viral Proteins; eIF-2 Kinase
PubMed: 34951963
DOI: 10.1016/j.molcel.2021.11.019 -
RNA Biology Jan 2023Precursor mRNA (pre-mRNA) splicing is an essential step in human gene expression and is carried out by a large macromolecular machine called the spliceosome. Given the... (Review)
Review
Precursor mRNA (pre-mRNA) splicing is an essential step in human gene expression and is carried out by a large macromolecular machine called the spliceosome. Given the spliceosome's role in shaping the cellular transcriptome, it is not surprising that mutations in the splicing machinery can result in a range of human diseases and disorders (spliceosomopathies). This review serves as an introduction into the main features of the pre-mRNA splicing machinery in humans and how changes in the function of its components can lead to diseases ranging from blindness to cancers. Recently, several drugs have been developed that interact directly with this machinery to change splicing outcomes at either the single gene or transcriptome-scale. We discuss the mechanism of action of several drugs that perturb splicing in unique ways. Finally, we speculate on what the future may hold in the emerging area of spliceosomopathies and spliceosome-targeted treatments.
Topics: Humans; RNA Precursors; RNA Splicing; Spliceosomes; Neoplasms
PubMed: 37528617
DOI: 10.1080/15476286.2023.2239601 -
Cell Jun 2021The N-methyladenosine (mA) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse...
The N-methyladenosine (mA) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an mA mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an mA-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site mA is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site mA is an ancient mechanism for splicing regulation.
Topics: Adenosine; Amino Acid Sequence; Animals; Base Sequence; Caenorhabditis elegans; Conserved Sequence; Diet; HeLa Cells; Humans; Introns; Methionine Adenosyltransferase; Methylation; Methyltransferases; Mice; Mutation; Nucleic Acid Conformation; Protein Binding; RNA Precursors; RNA Splice Sites; RNA Splicing; RNA, Messenger; RNA, Small Nuclear; S-Adenosylmethionine; Splicing Factor U2AF; Transcriptome
PubMed: 33930289
DOI: 10.1016/j.cell.2021.03.062 -
Molecular Cell May 2021O-linked β-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and...
O-linked β-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a "reader" of an O-GlcNAcylated NLS.
Topics: Active Transport, Cell Nucleus; Animals; Breast Neoplasms; Cell Proliferation; Female; Glucose; Glycosylation; HEK293 Cells; Humans; Lipogenesis; MCF-7 Cells; Mice, Nude; N-Acetylglucosaminyltransferases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; RNA Precursors; RNA Splicing; RNA, Messenger; Signal Transduction; Tumor Burden; alpha Karyopherins; beta Karyopherins; Mice
PubMed: 33657401
DOI: 10.1016/j.molcel.2021.02.009 -
Cell May 2021The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the...
The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.
Topics: Exons; Humans; Introns; RNA Precursors; RNA Splice Sites; RNA Splicing; RNA, Messenger; Spliceosomes; Transcription, Genetic; Transcriptome
PubMed: 33979654
DOI: 10.1016/j.cell.2021.04.012