RESUMO
The number of sequenced viral genomes has surged recently, presenting an opportunity to understand viral diversity and uncover unknown regulatory mechanisms. Here, we conducted a screening of 30,367 viral segments from 143 species representing 96 genera and 37 families. Using a library of viral segments in 3' UTR, we identified hundreds of elements impacting RNA abundance, translation, and nucleocytoplasmic distribution. To illustrate the power of this approach, we investigated K5, an element conserved in kobuviruses, and found its potent ability to enhance mRNA stability and translation in various contexts, including adeno-associated viral vectors and synthetic mRNAs. Moreover, we identified a previously uncharacterized protein, ZCCHC2, as a critical host factor for K5. ZCCHC2 recruits the terminal nucleotidyl transferase TENT4 to elongate poly(A) tails with mixed sequences, delaying deadenylation. This study provides a unique resource for virus and RNA research and highlights the potential of the virosphere for biological discoveries.
Assuntos
RNA , Sequências Reguladoras de Ácido Nucleico , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência de Bases , Proteínas/genética , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade de RNA , RNA Viral/genética , RNA Viral/metabolismoRESUMO
SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3' tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.
Assuntos
Betacoronavirus/genética , RNA Viral/genética , Transcriptoma , Animais , Chlorocebus aethiops , Epigênese Genética , Processamento Pós-Transcricional do RNA , SARS-CoV-2 , Análise de Sequência de RNA , Células VeroRESUMO
RNA tailing, or the addition of non-templated nucleotides to the 3' end of RNA, is the most frequent and conserved type of RNA modification. The addition of tails and their composition reflect RNA maturation stages and have important roles in determining the fate of the modified RNAs. Apart from canonical poly(A) polymerases, which add poly(A) tails to mRNAs in a transcription-coupled manner, a family of terminal nucleotidyltransferases (TENTs), including terminal uridylyltransferases (TUTs), modify RNAs post-transcriptionally to control RNA stability and activity. The human genome encodes 11 different TENTs with distinct substrate specificity, intracellular localization and tissue distribution. In this Review, we discuss recent advances in our understanding of non-canonical RNA tails, with a focus on the functions of human TENTs, which include uridylation, mixed tailing and post-transcriptional polyadenylation of mRNAs, microRNAs and other types of non-coding RNA.
Assuntos
Regulação da Expressão Gênica/fisiologia , Processamento Pós-Transcricional do RNA/fisiologia , RNA/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/fisiologia , Animais , Regulação da Expressão Gênica/genética , Humanos , MicroRNAs/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Poliadenilação , RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genéticaRESUMO
mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
Assuntos
RNA Mensageiro , Proteínas de Ligação a RNA , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Ligação Proteica , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Células HeLa , Fatores de Tempo , Aprendizado de MáquinaRESUMO
MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.
Assuntos
MicroRNAs , Processamento Pós-Transcricional do RNA , Humanos , MicroRNAs/metabolismo , RNA Interferente Pequeno , Ribonuclease III/genéticaRESUMO
MicroRNA maturation is initiated by RNase III DROSHA that cleaves the stem loop of primary microRNA. DROSHA functions together with its cofactor DGCR8 in a heterotrimeric complex known as Microprocessor. Here, we report the X-ray structure of DROSHA in complex with the C-terminal helix of DGCR8. We find that DROSHA contains two DGCR8-binding sites, one on each RNase III domain (RIIID), which mediate the assembly of Microprocessor. The overall structure of DROSHA is surprisingly similar to that of Dicer despite no sequence homology apart from the C-terminal part, suggesting that DROSHA may have evolved from a Dicer homolog. DROSHA exhibits unique features, including non-canonical zinc-finger motifs, a long insertion in the first RIIID, and the kinked link between Connector helix and RIIID, which explains the 11-bp-measuring "ruler" activity of DROSHA. Our study implicates the evolutionary origin of DROSHA and elucidates the molecular basis of Microprocessor assembly and primary microRNA processing.
Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/química , Sequência de Aminoácidos , Cristalografia por Raios X , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Evolução Molecular , Humanos , MicroRNAs/química , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.
Assuntos
MicroRNAs , Ribonuclease III , Animais , Humanos , Íntrons/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Prolina/genética , Prolina/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Processamento Pós-Transcricional do RNA , Peixe-ZebraRESUMO
MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an â¼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
Assuntos
MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/química , Ribonuclease III/química , Sequência de Bases , Dimerização , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Motivos de Nucleotídeos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
Although more than 100 types of RNA modification have been described thus far, most of them were thought to be rare in mRNAs and in regulatory noncoding RNAs. Recent developments have unveiled that at least some of the modifications are considerably abundant and widely conserved. This Minireview summarizes the molecular machineries and biological functions of methylation (N6-methyladenosine, m(6)A) and uridylation (U-tail).
Assuntos
Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Animais , Humanos , Metilação , MicroRNAs/metabolismo , Nucleotidiltransferases/metabolismo , Uridina Monofosfato/metabolismoRESUMO
Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than â¼ 25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , Células HeLa , Humanos , MicroRNAs/metabolismo , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Uridina Monofosfato/metabolismoRESUMO
RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. 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 loop3-11. 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-miRNA3-6. 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.
Assuntos
RNA Helicases DEAD-box , MicroRNAs , Conformação de Ácido Nucleico , Precursores de RNA , RNA Interferente Pequeno , Ribonuclease III , Humanos , Pareamento de Bases , RNA Helicases DEAD-box/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Precursores de RNA/biossíntese , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sequência de BasesRESUMO
Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.
Assuntos
Microscopia Crioeletrônica , RNA Helicases DEAD-box , MicroRNAs , Precursores de RNA , Ribonuclease III , Humanos , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/ultraestrutura , MicroRNAs/biossíntese , MicroRNAs/química , MicroRNAs/metabolismo , MicroRNAs/ultraestrutura , Mutação , Ribonuclease III/química , Ribonuclease III/genética , Ribonuclease III/metabolismo , Ribonuclease III/ultraestrutura , Precursores de RNA/química , Precursores de RNA/metabolismo , Precursores de RNA/ultraestrutura , RNA de Cadeia Dupla/metabolismo , Especificidade por SubstratoRESUMO
Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
Assuntos
MicroRNAs/genética , Processamento Pós-Transcricional do RNA/genética , Ribonuclease III/genética , Fatores de Processamento de Serina-Arginina/genética , Sítios de Ligação/genética , Genoma Humano/genética , Células HEK293 , Humanos , Interferência de RNARESUMO
SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.
Assuntos
Autoantígenos/genética , COVID-19/genética , RNA Viral/genética , Ribonucleoproteínas/genética , SARS-CoV-2/genética , COVID-19/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/patogenicidade , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Proteínas de Ligação a RNA/genética , SARS-CoV-2/patogenicidade , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Replicação Viral/genética , Antígeno SS-BRESUMO
Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.
Assuntos
Genômica , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Núcleo Celular/genética , Humanos , Processamento Pós-Transcricional do RNA/genéticaRESUMO
Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may change depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find miR-324 to be one of the strongly regulated miRNAs by arm switching and identify the terminal uridylyl transferases TUT4 and TUT7 to be the key regulators. Uridylation of pre-miR-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated, whereas the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.
Assuntos
MicroRNAs/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Camundongos , MicroRNAs/genética , Cultura Primária de Células , RNA Nucleotidiltransferases/metabolismo , Ribonuclease III/metabolismoRESUMO
RNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3' overhang. Dicer recognizes the 2 nt 3' overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1 nt) 3' overhang from Drosha processing and therefore require a 3'-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs act specifically on dsRNAs with a 1 nt 3' overhang, thereby creating a 2 nt 3' overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Nucleotidiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , Uridina Monofosfato/metabolismo , Sequência de Bases , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNARESUMO
LIN28 plays a critical role in developmental transition, glucose metabolism, and tumorigenesis. At the molecular level, LIN28 is known to repress maturation of let-7 microRNAs and enhance translation of certain mRNAs. In this study, we obtain a genome-wide view of the molecular function of LIN28A in mouse embryonic stem cells by carrying out RNA crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome footprinting. We find that, in addition to let-7 precursors, LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendoplasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins. Our study suggests a selective regulatory mechanism for ER-associated translation and reveals an unexpected role of LIN28A as a global suppressor of genes in the secretory pathway.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação/métodos , Camundongos , MicroRNAs/metabolismo , Ribossomos/metabolismo , Via Secretória , Análise de Sequência de RNARESUMO
Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing â¼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.
Assuntos
MicroRNAs/metabolismo , Motivos de Nucleotídeos , Processamento Pós-Transcricional do RNA , Ribonuclease III/metabolismo , Células HCT116 , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are â¼22 nucleotides in length, and they are produced by two RNase III proteins--Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.