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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 -
Molecular Cell Aug 2018Covalently closed circular RNAs (circRNAs) are produced by precursor mRNA back-splicing of exons of thousands of genes in eukaryotes. circRNAs are generally expressed at... (Review)
Review
Covalently closed circular RNAs (circRNAs) are produced by precursor mRNA back-splicing of exons of thousands of genes in eukaryotes. circRNAs are generally expressed at low levels and often exhibit cell-type-specific and tissue-specific patterns. Recent studies have shown that their biogenesis requires spliceosomal machinery and can be modulated by both cis complementary sequences and protein factors. The functions of most circRNAs remain largely unexplored, but known functions include sequestration of microRNAs or proteins, modulation of transcription and interference with splicing, and even translation to produce polypeptides. However, challenges exist at multiple levels to understanding of the regulation of circRNAs because of their circular conformation and sequence overlap with linear mRNA counterparts. In this review, we survey the recent progress on circRNA biogenesis and function and discuss technical obstacles in circRNA studies.
Topics: Alternative Splicing; Animals; Exons; Humans; MicroRNAs; RNA; RNA Precursors; RNA Splicing; RNA, Circular; RNA, Messenger; Spliceosomes
PubMed: 30057200
DOI: 10.1016/j.molcel.2018.06.034 -
Annual Review of Biochemistry 2015Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the... (Review)
Review
Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA-protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA-RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.
Topics: Alternative Splicing; Animals; Disease; Gene Expression Regulation; Humans; Mutation; RNA Precursors; RNA Splicing; RNA, Catalytic; Ribonucleoproteins, Small Nuclear; Spliceosomes
PubMed: 25784052
DOI: 10.1146/annurev-biochem-060614-034316 -
RNA (New York, N.Y.) Jan 2019MicroRNAs (miRNAs) are important regulators of gene expression that bind complementary target mRNAs and repress their expression. Precursor miRNA molecules undergo... (Review)
Review
MicroRNAs (miRNAs) are important regulators of gene expression that bind complementary target mRNAs and repress their expression. Precursor miRNA molecules undergo nuclear and cytoplasmic processing events, carried out by the endoribonucleases DROSHA and DICER, respectively, to produce mature miRNAs that are loaded onto the RISC (RNA-induced silencing complex) to exert their biological function. Regulation of mature miRNA levels is critical in development, differentiation, and disease, as demonstrated by multiple levels of control during their biogenesis cascade. Here, we will focus on post-transcriptional mechanisms and will discuss the impact of -acting sequences in precursor miRNAs, as well as -acting factors that bind to these precursors and influence their processing. In particular, we will highlight the role of general RNA-binding proteins (RBPs) as factors that control the processing of specific miRNAs, revealing a complex layer of regulation in miRNA production and function.
Topics: Animals; Cell Nucleus; Cytoplasm; Genetic Variation; Humans; MicroRNAs; RNA Precursors; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; RNA-Induced Silencing Complex; Ribonuclease III
PubMed: 30333195
DOI: 10.1261/rna.068692.118 -
RNA Biology 2015Unlike linear RNAs terminated with 5' caps and 3' tails, circular RNAs are characterized by covalently closed loop structures with neither 5' to 3' polarity nor... (Review)
Review
Unlike linear RNAs terminated with 5' caps and 3' tails, circular RNAs are characterized by covalently closed loop structures with neither 5' to 3' polarity nor polyadenylated tail. This intrinsic characteristic has led to the general under-estimation of the existence of circular RNAs in previous polyadenylated transcriptome analyses. With the advent of specific biochemical and computational approaches, a large number of circular RNAs from back-spliced exons (circRNAs) have been identified in various cell lines and across different species. Recent studies have uncovered that back-splicing requires canonical spliceosomal machinery and can be facilitated by both complementary sequences and specific protein factors. In this review, we highlight our current understanding of the regulation of circRNA biogenesis, including both the competition between splicing and back-splicing and the previously under-appreciated alternative circularization.
Topics: Humans; RNA; RNA Precursors; RNA Splicing; RNA, Circular; Regulatory Sequences, Nucleic Acid
PubMed: 25746834
DOI: 10.1080/15476286.2015.1020271 -
Nature Reviews. Molecular Cell Biology Jan 2017Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3' termini on mRNAs and other RNA polymerase II transcripts. It is widespread... (Review)
Review
Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3' termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation.
Topics: 3' Untranslated Regions; Active Transport, Cell Nucleus; Exons; Gene Expression Regulation; Humans; Polyadenylation; RNA Precursors; RNA Stability; RNA, Messenger; RNA-Binding Proteins; Ribonucleoprotein, U1 Small Nuclear
PubMed: 27677860
DOI: 10.1038/nrm.2016.116 -
Nature Reviews. Genetics Apr 2023The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the... (Review)
Review
The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the numerous pathologies induced or modified by splicing alterations. Major recent advances have been made in understanding the structures and functions of the splicing machinery, in the description and classification of physiological and pathological isoforms and in the development of the first therapies for genetic diseases based on modulation of splicing. Here, we review this progress and discuss important remaining challenges, including predicting splice sites from genomic sequences, understanding the variety of molecular mechanisms and logic of splicing regulation, and harnessing this knowledge for probing gene function and disease aetiology and for the design of novel therapeutic approaches.
Topics: RNA Precursors; RNA Splicing; Alternative Splicing; Introns
PubMed: 36526860
DOI: 10.1038/s41576-022-00556-8 -
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 -
Science (New York, N.Y.) Jan 2021To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA...
To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo-electron microscopy structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5' splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5' splice site, leading to the formation of a "growing intron loop." Loop formation may facilitate scanning of nascent pre-mRNA for the 3' splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.
Topics: Alternative Splicing; Animals; Cryoelectron Microscopy; Humans; Introns; Nucleic Acid Conformation; Protein Binding; Protein Domains; RNA Polymerase II; RNA Precursors; RNA, Messenger; Ribonucleoprotein, U1 Small Nuclear; Spliceosomes; Sus scrofa; Transcription, Genetic
PubMed: 33446560
DOI: 10.1126/science.abf1870 -
Nature Mar 2023RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs). However,...
RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.
Topics: Humans; Base Pairing; DEAD-box RNA Helicases; MicroRNAs; Ribonuclease III; RNA Interference; RNA, Double-Stranded; RNA, Small Interfering; RNA Precursors; Base Sequence; Nucleic Acid Conformation
PubMed: 36813957
DOI: 10.1038/s41586-023-05722-4