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Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.
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INTRODUCTION: Assessment of disease severity of vitiligo is exigent as it is a psychosomatic ailment. VIDA (vitiligo disease activity score) and VASI (vitiligo area severity index) were previously used for this evaluation. Recently, the introduction of two vitiligo specific tools, vitiligo impact scale (VIS)-22 and Vitiligo Quality of Life Index (VitiQoL) has aided in assessing the quality of life (QOL) in a pertinent manner. OBJECTIVES: To measure the QOL in vitiligo using disease specific indices (VitiQoL and VIS-22), to assess their relationship with disease severity (VASI and VIDA) and to determine the correlation between QOL scores (VIS-22 and VitiQoL). METHODS: This observational cross-sectional study included 195 patients with vitiligo, and their disease severity was calculated using VASI and VIDA scoring. Patients were asked to fill questionnaires for assessing the QOL using validated tools i.e. VIS-22 and VitiQoL. RESULTS: Significant correlation was demonstrated between both QOL scores and VASI score (P value 0.001) with slightly higher values for VitiQoL (r = 0.824) than with VIS 22 (r = 0.693). Both scores exhibited a significant association with VIDA score (P value < 0.001). Moreover, statistically significant correlation was found between VIS-22 and VitiQoL, thereby proving the concordance between these scores. CONCLUSIONS: The study infers that QOL seemed to be remarkably dependent on the clinical severity scores and that higher disease activity corresponds to poorer QOL. It is imperative to precisely assess burden of vitiligo and the impairments caused by it in order to aid multi-modality management and allow more standardized research.
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Nonsense-mediated mRNA decay (NMD) is a quality control pathway in eukaryotes that continuously monitors mRNA transcripts to ensure truncated polypeptides are not produced. The expression of many normal mRNAs that encode full-length polypeptides is also regulated by this pathway. Such transcript surveillance by NMD is intimately linked to translation termination. When a ribosome terminates translation at a normal termination codon, NMD is not activated, and mRNA can undergo repeated rounds of translation. On the other hand, when translation termination is deemed abnormal, such as that on a premature termination codon, it leads to a series of poorly understood events involving the NMD pathway, which destabilizes the transcript. In this review, we summarize our current understanding of how the NMD machinery interfaces with the translation termination factors to initiate NMD. We also discuss a variety of cis-acting sequence contexts and trans-acting factors that can cause readthrough, ribosome reinitiation, or ribosome frameshifting at stop codons predicted to induce NMD. These alternative outcomes can lead to the ribosome translating downstream of such stop codons and hence the transcript escaping NMD. NMD escape via these mechanisms can have wide-ranging implications on human health, from being exploited by viruses to hijack host cell systems to being harnessed as potential therapeutic possibilities to treat genetic diseases.
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Degradação do RNAm Mediada por Códon sem Sentido , Ribossomos , Humanos , Códon de Terminação/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Nonsense-mediated mRNA decay (NMD) is governed by the three conserved factors-UPF1, UPF2, and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B-dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B-dependent and -independent manner. While UPF3A is almost dispensable for NMD in wild-type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC-binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved "mid" domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.
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Degradação do RNAm Mediada por Códon sem Sentido , Proteínas de Ligação a RNA , Éxons , Células HCT116 , Humanos , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
RNA-binding proteins (RBPs) regulate all aspects of RNA metabolism. The ability to identify RNA targets bound by RBPs is critical for understanding RBP function. While powerful techniques are available to identify binding sites of individual RBPs at high resolution, it remains challenging to unravel binding sites of multicomponent ribonucleoproteins (RNPs) where multiple RBPs or proteins function cooperatively to bind to target RNAs. To fill this gap, we have previously developed RNA Immunoprecipitation in Tandem followed by high-throughput sequencing (RIPiT-seq) to characterize RNA targets of compositionally distinct RNP complexes by sequentially immunoprecipitating two proteins from the same RNP and sequencing the co-purifying RNA footprints. Here, we provide an updated and improved protocol for RIPiT-seq. In this protocol, we have used CRISPR-Cas9 to introduce affinity tag to endogenous protein of interest to capture a more representative state of an RNP complex. We present a modified protocol for library preparation for high-throughput sequencing so that it exclusively uses equipment and reagents available in a standard molecular biology lab. This updated custom library preparation protocol is compatible with commercial PCR multiplexing systems for Illumina sequencing platform for simultaneous and cost-effective analysis of large number of samples.
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RNA , Ribonucleoproteínas , Sítios de Ligação , Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação , RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Nonsense-mediated mRNA decay (NMD) is a conserved translation-coupled quality control mechanism in all eukaryotes that regulates the expression of a significant fraction of both the aberrant and normal transcriptomes. In vertebrates, NMD has become an essential process owing to expansion of the diversity of NMD-regulated transcripts, particularly during various developmental processes. Surprisingly, however, some core NMD factors that are essential for NMD in simpler organisms appear to be dispensable for vertebrate NMD. At the same time, numerous NMD enhancers and suppressors have been identified in multicellular organisms including vertebrates. Collectively, the available data suggest that vertebrate NMD is a complex, branched pathway wherein individual branches regulate specific mRNA subsets to fulfill distinct physiological functions.
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Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Animais , Humanos , Transcriptoma/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Many post-transcriptional mechanisms operate via mRNA 3'UTRs to regulate protein expression, and such controls are crucial for development. We show that homozygous mutations in two zebrafish exon junction complex (EJC) core genes rbm8a and magoh leads to muscle disorganization, neural cell death, and motor neuron outgrowth defects, as well as dysregulation of mRNAs subjected to nonsense-mediated mRNA decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction. Intriguingly, we find that EJC-dependent NMD also regulates a subset of transcripts that contain 3'UTR introns (3'UI) < 50 nts downstream of a stop codon. Some transcripts containing such stop codon-proximal 3'UI are also NMD-sensitive in cultured human cells and mouse embryonic stem cells. We identify 167 genes that contain a conserved proximal 3'UI in zebrafish, mouse and humans. foxo3b is one such proximal 3'UI-containing gene that is upregulated in zebrafish EJC mutant embryos, at both mRNA and protein levels, and loss of foxo3b function in EJC mutant embryos significantly rescues motor axon growth defects. These data are consistent with EJC-dependent NMD regulating foxo3b mRNA to control protein expression during zebrafish development. Our work shows that the EJC is critical for normal zebrafish development and suggests that proximal 3'UIs may serve gene regulatory function in vertebrates.
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Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Neurogênese/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Códon de Terminação , Conjuntos de Dados como Assunto , Embrião não Mamífero , Éxons/genética , Redes Reguladoras de Genes/genética , Homozigoto , Humanos , Íntrons/genética , Camundongos , Músculo Esquelético/inervação , Mutagênese , Mutação , Crescimento Neuronal/genética , Proteínas Nucleares/genética , Terminação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Alinhamento de Sequência , Regulação para Cima , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion.
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Sítios de Ligação/genética , Ligação Proteica/genética , RNA/química , RNA/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Linhagem Celular , Células Eucarióticas/metabolismo , Éxons/genética , Células HEK293 , Humanos , Imunoprecipitação/métodos , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.
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Leucemia Mieloide/genética , Mutação , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , RNA Ribossômico/genética , Transcrição Gênica , Doença Aguda , Animais , Linhagem Celular Tumoral , Proliferação de Células , Células HEK293 , Humanos , Células K562 , Leucemia Mieloide/patologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Nucleofosmina , Biossíntese de Proteínas/genética , Células THP-1 , Transplante HeterólogoRESUMO
RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT's advantages and applications and discuss some of its limitations.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Células HEK293 , Humanos , Reação em Cadeia da PolimeraseRESUMO
The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition, and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that the EJC originates as an SR-rich mega-dalton-sized RNP that contains RNPS1 but lacks CASC3. Sometime before or during translation, the EJC undergoes compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general, whereas CASC3 is needed for NMD of only select mRNAs. The switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.
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Éxons/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Ribonucleoproteínas/química , Animais , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Hidrodinâmica , Cinética , Camundongos , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.
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Proteínas Argonautas/metabolismo , Clonagem Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Pequeno RNA não Traduzido/genética , Biologia Computacional/métodos , Interpretação Estatística de Dados , Biblioteca Gênica , Pequeno RNA não Traduzido/isolamento & purificação , Pequeno RNA não Traduzido/metabolismo , Análise de Sequência de RNARESUMO
During messenger RNA (mRNA) biogenesis and processing in the nucleus, many proteins are imprinted on mRNAs assembling them into messenger ribonucleoproteins (mRNPs). Some of these proteins remain stably bound within mRNPs and have a long-lasting impact on their fate. One of the best-studied examples is the exon junction complex (EJC), a multiprotein complex deposited primarily 24 nucleotides upstream of exon-exon junctions as a consequence of pre-mRNA splicing. The EJC maintains a stable, sequence-independent, hold on the mRNA until its removal during translation in the cytoplasm. Acting as a molecular shepherd, the EJC travels with mRNA across the cellular landscape coupling pre-mRNA splicing to downstream, posttranscriptional processes such as mRNA export, mRNA localization, translation, and nonsense-mediated mRNA decay (NMD). In this review, we discuss our current understanding of the EJC's functions during these processes, and expound its newly discovered functions (e.g., pre-mRNA splicing). Another focal point is the recently unveiled in vivo EJC interactome, which has shed new light on the EJC's location on the spliced RNAs and its intimate relationship with other mRNP components. We summarize new strides being made in connecting the EJC's molecular function with phenotypes, informed by studies of human disorders and model organisms. The progress toward understanding EJC functions has revealed, in its wake, even more questions, which are discussed throughout. WIREs RNA 2017, 8:e1411. doi: 10.1002/wrna.1411 For further resources related to this article, please visit the WIREs website.
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Éxons/genética , Splicing de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Humanos , RNA Mensageiro/genética , Ribonucleoproteínas/genéticaRESUMO
Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: Genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5' proximal-intron-minus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the exon junction complex (EJC) at noncanonical 5' proximal positions. Finally, N1-methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising â¼20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N1-methyladenosines in the early coding region, and enrichment for noncanonical binding by the EJC.
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Regiões 5' não Traduzidas , Adenosina/análogos & derivados , Sequência de Bases , Íntrons , Biossíntese de Proteínas , Deleção de Sequência , Adenosina/genética , Adenosina/metabolismo , Códon de Iniciação/química , Códon de Iniciação/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Éxons , Humanos , Fases de Leitura Aberta , Ligação Proteica , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Throughout their lifetimes, messenger RNAs (mRNAs) associate with proteins to form ribonucleoproteins (mRNPs). Since the discovery of the first mRNP component more than 40 years ago, what is known as the mRNA interactome now comprises >1,000 proteins. These proteins bind mRNAs in myriad ways with varying affinities and stoichiometries, with many assembling onto nascent RNAs in a highly ordered process during transcription and precursor mRNA (pre-mRNA) processing. The nonrandom distribution of major mRNP proteins observed in transcriptome-wide studies leads us to propose that mRNPs are organized into three major domains loosely corresponding to 5' untranslated regions (UTRs), open reading frames, and 3' UTRs. Moving from the nucleus to the cytoplasm, mRNPs undergo extensive remodeling as they are first acted upon by the nuclear pore complex and then by the ribosome. When not being actively translated, cytoplasmic mRNPs can assemble into large multi-mRNP assemblies or be permanently disassembled and degraded. In this review, we aim to give the reader a thorough understanding of past and current eukaryotic mRNP research.
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Ribonucleoproteínas/química , Transporte Ativo do Núcleo Celular , Animais , Humanos , Biossíntese de Proteínas , Splicing de RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3' untranslated regions (UTRs) or in 'strongly distal' 3' UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3' UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins.
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Proteínas do Citoesqueleto/metabolismo , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Proteínas de Ligação a RNA/genéticaRESUMO
Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes. RNA:protein immunoprecipitation in tandem (RIPiT) yields highly specific RNA footprints of cellular RNPs isolated via two sequential purifications; the resulting RNA footprints can then be identified by high-throughput sequencing (Seq). RIPiT-Seq is broadly applicable to all RBPs regardless of their RNA binding mode and thus provides a means to map the RNA binding sites of RBPs with poor inherent ultraviolet (UV) crosslinkability. Further, among current high-throughput approaches, RIPiT has the unique capacity to differentiate binding sites of RNPs with overlapping protein composition. It is therefore particularly suited for studying dynamic RNP assemblages whose composition evolves as gene expression proceeds.
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Pegadas de Proteínas/métodos , RNA/química , Proteínas Recombinantes de Fusão/química , Ribonucleoproteínas/química , Transcriptoma , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Formaldeído/química , Regulação da Expressão Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Modelos Moleculares , Ligação Proteica , RNA/genética , Proteínas Recombinantes de Fusão/genética , Ribonucleoproteínas/genéticaRESUMO
SUMMARY: Unlike DNA, RNA abundances can vary over several orders of magnitude. Thus, identification of RNA-protein binding sites from high-throughput sequencing data presents unique challenges. Although peak identification in ChIP-Seq data has been extensively explored, there are few bioinformatics tools tailored for peak calling on analogous datasets for RNA-binding proteins. Here we describe ASPeak (abundance sensitive peak detection algorithm), an implementation of an algorithm that we previously applied to detect peaks in exon junction complex RNA immunoprecipitation in tandem experiments. Our peak detection algorithm yields stringent and robust target sets enabling sensitive motif finding and downstream functional analyses. AVAILABILITY: ASPeak is implemented in Perl as a complete pipeline that takes bedGraph files as input. ASPeak implementation is freely available at https://sourceforge.net/projects/as-peak under the GNU General Public License. ASPeak can be run on a personal computer, yet is designed to be easily parallelizable. ASPeak can also run on high performance computing clusters providing efficient speedup. The documentation and user manual can be obtained from http://master.dl.sourceforge.net/project/as-peak/manual.pdf.