RESUMEN
Translational regulation in tissue environments during in vivo viral pathogenesis has rarely been studied due to the lack of translatomes from virus-infected tissues, although a series of translatome studies using in vitro cultured cells with viral infection have been reported. In this study, we exploited tissue-optimized ribosome profiling (Ribo-seq) and severe-COVID-19 model mice to establish the first temporal translation profiles of virus and host genes in the lungs during SARS-CoV-2 pathogenesis. Our datasets revealed not only previously unknown targets of translation regulation in infected tissues but also hitherto unreported molecular signatures that contribute to tissue pathology after SARS-CoV-2 infection. Specifically, we observed gradual increases in pseudoribosomal ribonucleoprotein (RNP) interactions that partially overlapped the trails of ribosomes, being likely involved in impeding translation elongation. Contemporaneously developed ribosome heterogeneity with predominantly dysregulated 5 S rRNP association supported the malfunction of elongating ribosomes. Analyses of canonical Ribo-seq reads (ribosome footprints) highlighted two obstructive characteristics to host gene expression: ribosome stalling on codons within transmembrane domain-coding regions and compromised translation of immunity- and metabolism-related genes with upregulated transcription. Our findings collectively demonstrate that the abrogation of translation integrity may be one of the most critical factors contributing to pathogenesis after SARS-CoV-2 infection of tissues.
Asunto(s)
COVID-19 , Animales , Ratones , ARN Mensajero/genética , COVID-19/genética , SARS-CoV-2/genética , Biosíntesis de Proteínas , Pulmón/metabolismoRESUMEN
Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs); RNA1.2, RNA2.7, RNA4.9, and RNA5.0. Little is known about the function of these lncRNAs in the virus life cycle. Here, we dissected the functional and molecular landscape of HCMV lncRNAs. We found that HCMV lncRNAs occupy ~ 30% and 50-60% of total and poly(A)+viral transcriptome, respectively, throughout virus life cycle. RNA1.2, RNA2.7, and RNA4.9, the three abundantly expressed lncRNAs, appear to be essential in all infection states. Among these three lncRNAs, depletion of RNA2.7 and RNA4.9 results in the greatest defect in maintaining latent reservoir and promoting lytic replication, respectively. Moreover, we delineated the global post-transcriptional nature of HCMV lncRNAs by nanopore direct RNA sequencing and interactome analysis. We revealed that the lncRNAs are modified with N6-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth analysis demonstrated that m6A machineries stabilize HCMV lncRNAs, which could account for the overwhelming abundance of viral lncRNAs. Our study lays the groundwork for understanding the viral lncRNA-mediated regulation of host-virus interaction throughout the HCMV life cycle.
Asunto(s)
Infecciones por Citomegalovirus , ARN Largo no Codificante , Humanos , Citomegalovirus/genética , ARN Largo no Codificante/genética , Células Cultivadas , Transcriptoma , Replicación Viral/genéticaRESUMEN
RNA modifications are a common occurrence across all domains of life. Several chemical modifications, including N6-methyladenosine, have also been found in viral transcripts and viral RNA genomes. Some of the modifications increase the viral replication efficiency while also helping the virus to evade the host immune system. Nonetheless, there are numerous examples in which the host's RNA modification enzymes function as antiviral factors. Although established methods like MeRIP-seq and miCLIP can provide a transcriptome- wide overview of how viral RNA is modified, it is difficult to distinguish between the complex overlapping viral transcript isoforms using the short read-based techniques. Nanopore direct RNA sequencing (DRS) provides both long reads and direct signal readings, which may carry information about the modifications. Here, we describe a refined protocol for analyzing the RNA modifications in viral transcriptomes using nanopore technology.
Asunto(s)
Secuenciación de Nanoporos , Nanoporos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Viral/genética , Análisis de Secuencia de ARN/métodos , TranscriptomaRESUMEN
The accuracy of methods for assembling transcripts from short-read RNA sequencing data is limited by the lack of long-range information. Here we introduce Ladder-seq, an approach that separates transcripts according to their lengths before sequencing and uses the additional information to improve the quantification and assembly of transcripts. Using simulated data, we show that a kallisto algorithm extended to process Ladder-seq data quantifies transcripts of complex genes with substantially higher accuracy than conventional kallisto. For reference-based assembly, a tailored scheme based on the StringTie2 algorithm reconstructs a single transcript with 30.8% higher precision than its conventional counterpart and is more than 30% more sensitive for complex genes. For de novo assembly, a similar scheme based on the Trinity algorithm correctly assembles 78% more transcripts than conventional Trinity while improving precision by 78%. In experimental data, Ladder-seq reveals 40% more genes harboring isoform switches compared to conventional RNA sequencing and unveils widespread changes in isoform usage upon m6A depletion by Mettl14 knockout.
Asunto(s)
ARN , Transcriptoma , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Isoformas de Proteínas , RNA-Seq , Análisis de Secuencia de ARN/métodos , Transcriptoma/genéticaRESUMEN
TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.
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Citomegalovirus/genética , Virus de la Hepatitis B/genética , Interacciones Huésped-Patógeno/fisiología , ARN Viral/metabolismo , Línea Celular , Citomegalovirus/patogenicidad , Virus de la Hepatitis B/patogenicidad , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Filogenia , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , ARN Viral/químicaRESUMEN
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.
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Betacoronavirus/genética , ARN Viral/genética , Transcriptoma , Animales , Chlorocebus aethiops , Epigénesis Genética , Procesamiento Postranscripcional del ARN , SARS-CoV-2 , Análisis de Secuencia de ARN , Células VeroRESUMEN
The severe acute respiratory coronavirus 2 (SARS-CoV-2), which emerged in December 2019 in Wuhan, China, has spread rapidly to over a dozen countries. Especially, the spike of case numbers in South Korea sparks pandemic worries. This virus is reported to spread mainly through person-to-person contact via respiratory droplets generated by coughing and sneezing, or possibly through surface contaminated by people coughing or sneezing on them. More critically, there have been reports about the possibility of this virus to transmit even before a virus-carrying person to show symptoms. Therefore, a low-cost, easy-access protocol for early detection of this virus is desperately needed. Here, we have established a real-time reverse-transcription PCR (rtPCR)-based assay protocol composed of easy specimen self-collection from a subject via pharyngeal swab, Trizol-based RNA purification, and SYBR Green-based rtPCR. This protocol shows an accuracy and sensitivity limit of 1-10 virus particles as we tested with a known lentivirus. The cost for each sample is estimated to be less than 15 US dollars. Overall time it takes for an entire protocol is estimated to be less than 4 hours. We propose a cost-effective, quick-and-easy method for early detection of SARS-CoV-2 at any conventional Biosafety Level II laboratories that are equipped with a rtPCR machine. Our newly developed protocol should be helpful for a first-hand screening of the asymptomatic virus-carriers for further prevention of transmission and early intervention and treatment for the rapidly propagating virus.
RESUMEN
MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification of miRNAs has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here, we quantify miRNAs and their variants (known as isomiRs) by an improved sRNA-seq protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which minimize the ligation bias during library preparation. Measurement using AQ-seq allows us to correct the previously misannotated 5' end usage and strand preference in public databases. Importantly, the analysis of 5' terminal heterogeneity reveals widespread alternative processing events which have been underestimated. We also identify highly uridylated miRNAs originating from the 3p strands, indicating regulations mediated by terminal uridylyl transferases at the pre-miRNA stage. Taken together, our study reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/genética , Interferencia de ARN , Secuencia de Bases , Inmunoprecipitación , Anotación de Secuencia Molecular/métodos , Polietilenglicoles/química , Análisis de Secuencia de ARNRESUMEN
RNA tails play integral roles in the regulation of messenger RNA (mRNA) translation and decay. Guanylation of the poly(A) tail was discovered recently, yet the enzymology and function remain obscure. Here we identify TENT4A (PAPD7) and TENT4B (PAPD5) as the enzymes responsible for mRNA guanylation. Purified TENT4 proteins generate a mixed poly(A) tail with intermittent non-adenosine residues, the most common of which is guanosine. A single guanosine residue is sufficient to impede the deadenylase CCR4-NOT complex, which trims the tail and exposes guanosine at the 3' end. Consistently, depletion of TENT4A and TENT4B leads to a decrease in mRNA half-life and abundance in cells. Thus, TENT4A and TENT4B produce a mixed tail that shields mRNA from rapid deadenylation. Our study unveils the role of mixed tailing and expands the complexity of posttranscriptional gene regulation.
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Proteínas Cromosómicas no Histona/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Regulación de la Expresión Génica , Procesamiento de Término de ARN 3' , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Cromosómicas no Histona/genética , ADN Polimerasa Dirigida por ADN/genética , Exorribonucleasas/metabolismo , Fibroblastos , Eliminación de Gen , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , ARN Nucleotidiltransferasas/genéticaRESUMEN
Multiple deadenylases are known in vertebrates, the PAN2-PAN3 (PAN2/3) and CCR4-NOT (CNOT) complexes, and PARN, yet their differential functions remain ambiguous. Moreover, the role of poly(A) binding protein (PABP) is obscure, limiting our understanding of the deadenylation mechanism. Here, we show that CNOT serves as a predominant nonspecific deadenylase for cytoplasmic poly(A)+ RNAs, and PABP promotes deadenylation while preventing premature uridylation and decay. PAN2/3 selectively trims long tails (>â¼150 nt) with minimal effect on transcriptome, whereas PARN does not affect mRNA deadenylation. CAF1 and CCR4, catalytic subunits of CNOT, display distinct activities: CAF1 trims naked poly(A) segments and is blocked by PABPC, whereas CCR4 is activated by PABPC to shorten PABPC-protected sequences. Concerted actions of CAF1 and CCR4 delineate the â¼27 nt periodic PABPC footprints along shortening tail. Our study unveils distinct functions of deadenylases and PABPC, re-drawing the view on mRNA deadenylation and regulation.
Asunto(s)
Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Receptores CCR4/metabolismo , Factores de Transcripción/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular/metabolismo , Citoplasma/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Poli A/metabolismo , Proteínas de Unión a Poli(A)/genética , Poliadenilación , ARN Mensajero/genética , Receptores CCR4/genética , Factores de Transcripción/genética , TranscriptomaRESUMEN
During the maternal-to-zygotic transition (MZT), maternal RNAs are actively degraded and replaced by newly synthesized zygotic transcripts in a highly coordinated manner. However, it remains largely unknown how maternal mRNA decay is triggered in early vertebrate embryos. Here, through genome-wide profiling of RNA abundance and 3' modification, we show that uridylation is induced at the onset of maternal mRNA clearance. The temporal control of uridylation is conserved in vertebrates. When the homologs of terminal uridylyltransferases TUT4 and TUT7 (TUT4/7) are depleted in zebrafish and Xenopus, maternal mRNA clearance is significantly delayed, leading to developmental defects during gastrulation. Short-tailed mRNAs are selectively uridylated by TUT4/7, with the highly uridylated transcripts degraded faster during the MZT than those with unmodified poly(A) tails. Our study demonstrates that uridylation plays a crucial role in timely mRNA degradation, thereby allowing the progression of early development.
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Embrión de Mamíferos/enzimología , Embrión no Mamífero/enzimología , Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Transcriptoma , Xenopus laevis/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Ratones Endogámicos ICR , Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
Eukaryotic mRNAs are subject to multiple types of tailing that critically influence mRNA stability and translatability. To investigate RNA tails at the genomic scale, we previously developed TAIL-seq, but its low sensitivity precluded its application to biological materials of minute quantity. In this study, we report a new version of TAIL-seq (mRNA TAIL-seq [mTAIL-seq]) with enhanced sequencing depth for mRNAs (by â¼1000-fold compared with the previous version). The improved method allows us to investigate the regulation of poly(A) tails in Drosophila oocytes and embryos. We found that maternal mRNAs are polyadenylated mainly during late oogenesis, prior to fertilization, and that further modulation occurs upon egg activation. Wispy, a noncanonical poly(A) polymerase, adenylates the vast majority of maternal mRNAs, with a few intriguing exceptions such as ribosomal protein transcripts. By comparing mTAIL-seq data with ribosome profiling data, we found a strong coupling between poly(A) tail length and translational efficiency during egg activation. Our data suggest that regulation of poly(A) tails in oocytes shapes the translatomic landscape of embryos, thereby directing the onset of animal development. By virtue of the high sensitivity, low cost, technical robustness, and broad accessibility, mTAIL-seq will be a potent tool to improve our understanding of mRNA tailing in diverse biological systems.
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Oocitos/metabolismo , Poli A/metabolismo , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodos , Animales , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Células HeLa , Humanos , Poli A/química , Poli A/genética , Poliadenilación , Polinucleotido Adenililtransferasa/metabolismo , ARN Mensajero/química , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Poly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell-cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is <20 nucleotides. However, as most genes have >20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle.
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Mitosis , Poli A/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Proteína Quinasa CDC2 , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Quinasas Ciclina-Dependientes/biosíntesis , Quinasas Ciclina-Dependientes/genética , ADN-Topoisomerasas de Tipo II/biosíntesis , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas F-Box/biosíntesis , Proteínas F-Box/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Biblioteca de Genes , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Poli A/genética , Poli dA-dT/genética , Poli dA-dT/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Estabilidad del ARN , ARN Mensajero/genética , Ribosomas/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Factores de TiempoRESUMEN
Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.
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Genoma , Interferencia de ARN , Animales , Inteligencia Artificial , Humanos , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
Temporal profiles of miRNA activity during productive virus infection can provide fundamental insights into host-virus interactions. Most reported miRNA targetome analyses in the context of virus infection have been performed in latently infected cells and lack reliable models for quantifying the suppression efficacy at specific miRNA target sites. Here, we identified highly competent temporal miRNA targetomes during lytic HCMV infection by using AGO-CLIP-seq together with a bioinformatic method that quantifies miRNA functionality at a specific target site, called ACE-scoring. The repression efficiency at target sites correlates with the magnitude of the ACE-score, and temporal HCMV-encoded miRNA targetomes identified by ACE-scoring were significantly enriched in functional categories involved in pathways central for HCMV biology. Furthermore, comparative analysis between human and viral miRNA targetomes supports the existence of intimate cooperation and co-targeting between them. Our holistic survey provides a valuable resource for understanding host-virus interactions during lytic HCMV infection.
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Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , MicroARNs , Citomegalovirus/patogenicidad , Perfilación de la Expresión Génica/métodos , Células HeLa/virología , Humanos , Interferones/metabolismo , Quinasas Janus/metabolismo , MicroARNs/genética , Reproducibilidad de los Resultados , Factores de Transcripción STAT/metabolismo , Análisis de Secuencia de ARN/métodos , Transducción de Señal/genéticaRESUMEN
Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3' overhang, TUT7 restores the canonical end structure (2-nt 3' overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3' end is further recessed into the stem (as in 3' trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.
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MicroARNs/metabolismo , ARN Nucleotidiltransferasas/fisiología , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Uridina Monofosfato/metabolismo , Nucleótidos de Adenina/metabolismo , Secuencia de Bases , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/metabolismo , Procesamiento Postranscripcional del ARN/genética , Estabilidad del ARN/genética , Nucleótidos de Uracilo/metabolismoRESUMEN
SUMMARY: Deep sequencing of small RNAs has become a routine process in recent years, but no dedicated viewer is as yet available to explore the sequence features simultaneously along with secondary structure and gene expression of microRNA (miRNA). We present a highly interactive application that visualizes the sequence alignment, secondary structure and normalized read counts in synchronous multipanel windows. This helps users to easily examine the relationships between the structure of precursor and the sequences and abundance of final products and thereby will facilitate the studies on miRNA biogenesis and regulation. The project manager handles multiple samples of multiple groups. The read alignment is imported in BAM file format. Implemented features comprise sorting, zooming, highlighting, editing, filtering, saving, exporting, etc. Currently, miRseqViewer supports 84 organisms whose annotation is available at miRBase. AVAILABILITY AND IMPLEMENTATION: miRseqViewer, implemented in Java, is available at https://github.com/insoo078/mirseqviewer or at http://msv.kobic.re.kr. CONTACT: sanghyuk@ewha.ac.kr.
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Biología Computacional/métodos , Gráficos por Computador , Bases de Datos de Ácidos Nucleicos , MicroARNs/genética , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Alineación de SecuenciaRESUMEN
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.