Precursor messenger RNA (pre-mRNA) splicing is an essential step in the flow of information from DNA to protein in all eukaryotes. Research over the past four decades has molecularly delineated the ...splicing pathway, including characterization of the detailed splicing reaction, definition of the spliceosome and identification of its components, and biochemical analysis of the various splicing complexes and their regulation. Structural information is central to mechanistic understanding of pre-mRNA splicing by the spliceosome. X-ray crystallography of the spliceosomal components and subcomplexes is complemented by electron microscopy of the intact spliceosome. In this Review, I discuss recent atomic-resolution structures of the intact spliceosome at different stages of the splicing cycle. These structures have provided considerable mechanistic insight into pre-mRNA splicing and have corroborated and explained a large body of genetic and biochemical data. Together, the structural data have proved that the spliceosome is a protein-directed metalloribozyme.
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.
CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and ...III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN-3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR-Cas systems so far described.
Genotoxic agents, that are used in cancer therapy, elicit the reprogramming of the transcriptome of cancer cells. These changes reflect the cellular response to stress and underlie some of the ...mechanisms leading to drug resistance. Here, we profiled genome-wide changes in pre-mRNA splicing induced by cisplatin in breast cancer cells. Among the set of cisplatin-induced alternative splicing events we focused on COASY, a gene encoding a mitochondrial enzyme involved in coenzyme A biosynthesis. Treatment with cisplatin induces the production of a short isoform of COASY lacking exons 4 and 5, whose depletion impedes mitochondrial function and decreases sensitivity to cisplatin. We identified RBM39 as a major effector of the cisplatin-induced effect on COASY splicing. RBM39 also controls a genome-wide set of alternative splicing events partially overlapping with the cisplatin-mediated ones. Unexpectedly, inactivation of RBM39 in response to cisplatin involves its interaction with the AP-1 family transcription factor c-Jun that prevents RBM39 binding to pre-mRNA. Our findings therefore uncover a novel cisplatin-induced interaction between a splicing regulator and a transcription factor that has a global impact on alternative splicing and contributes to drug resistance.
The synthesis of new ribosomes begins during transcription of the rRNA and is widely assumed to follow an orderly 5′ to 3′ gradient. To visualize co-transcriptional assembly of ribosomal protein-RNA ...complexes in real time, we developed a single-molecule platform that simultaneously monitors transcription and protein association with the elongating transcript. Unexpectedly, the early assembly protein uS4 binds newly made pre-16S rRNA only transiently, likely due to non-native folding of the rRNA during transcription. Stable uS4 binding became more probable only in the presence of additional ribosomal proteins that bind upstream and downstream of protein uS4 by allowing productive assembly intermediates to form earlier. We propose that dynamic sampling of elongating RNA by multiple proteins overcomes heterogeneous RNA folding, preventing assembly bottlenecks and initiating assembly within the transcription time window. This may be a common feature of transcription-coupled RNP assembly.
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•Single-molecule assay monitors transcription and RNP assembly in real time•Proteins sample the pre-rRNA as soon as the binding site is synthesized•Multiple ribosomal proteins commit the rRNA to assembly during transcription•All-or-none co-transcriptional RNP assembly mechanism avoids assembly pitfalls
Concomitant single-molecule detection of transcription elongation and protein recruitment reveals dynamic sampling of nascent pre-ribosomal RNA by ribosomal proteins until multiple proteins can bind and commit the elongating pre-rRNA to productive assembly.
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.
RNA G-quadruplexes (G4s) are formed by G-rich RNA sequences in protein-coding (mRNA) and non-coding (ncRNA) transcripts that fold into a four-stranded conformation. Experimental studies and ...bioinformatic predictions support the view that these structures are involved in different cellular functions associated to both DNA processes (telomere elongation, recombination and transcription) and RNA post-transcriptional mechanisms (including pre-mRNA processing, mRNA turnover, targeting and translation). An increasing number of different diseases have been associated with the inappropriate regulation of RNA G4s exemplifying the potential importance of these structures on human health. Here, we review the different molecular mechanisms underlying the link between RNA G4s and human diseases by proposing several overlapping models of deregulation emerging from recent research, including (i) sequestration of RNA-binding proteins, (ii) aberrant expression or localization of RNA G4-binding proteins, (iii) repeat associated non-AUG (RAN) translation, (iv) mRNA translational blockade and (v) disabling of protein-RNA G4 complexes. This review also provides a comprehensive survey of the functional RNA G4 and their mechanisms of action. Finally, we highlight future directions for research aimed at improving our understanding on RNA G4-mediated regulatory mechanisms linked to diseases.
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.
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•Visualizing the ultrastructure of FC/DFC and rDNA arrangements in human nucleoli•Processing factors, such as FBL, form protein clusters and then assemble into a DFC•Self-association of GAR in FBL ensures sorting and processing of nascent 47S pre-rRNA•Nascent pre-rRNA sorting via a phase-separation mechanism promotes DFC assembly
Yao et al. unveil the FC/DFC ultrastructure and rDNA arrangements in human nucleoli and show that a phase-separation mechanism promotes nascent pre-rRNA sorting and processing and the assembly of the DFC sub-nucleolar region.
Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 ...nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.
Abstract
MicroRNAs (miRNAs) related single-nucleotide variations (SNVs), including single-nucleotide polymorphisms (SNPs) and disease-related variations (DRVs) in miRNAs and miRNA-target binding ...sites, can affect miRNA functions and/or biogenesis, thus to impact on phenotypes. miRNASNP is a widely used database for miRNA-related SNPs and their effects. Here, we updated it to miRNASNP-v3 (http://bioinfo.life.hust.edu.cn/miRNASNP/) with tremendous number of SNVs and new features, especially the DRVs data. We analyzed the effects of 7 161 741 SNPs and 505 417 DRVs on 1897 pre-miRNAs (2630 mature miRNAs) and 3′UTRs of 18 152 genes. miRNASNP-v3 provides a one-stop resource for miRNA-related SNVs research with the following functions: (i) explore associations between miRNA-related SNPs/DRVs and diseases; (ii) browse the effects of SNPs/DRVs on miRNA-target binding; (iii) functional enrichment analysis of miRNA target gain/loss caused by SNPs/DRVs; (iv) investigate correlations between drug sensitivity and miRNA expression; (v) inquire expression profiles of miRNAs and their targets in cancers; (vi) browse the effects of SNPs/DRVs on pre-miRNA secondary structure changes; and (vii) predict the effects of user-defined variations on miRNA-target binding or pre-miRNA secondary structure. miRNASNP-v3 is a valuable and long-term supported resource in functional variation screening and miRNA function studies.
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