RESUMEN
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. The loss of TDRD5 and TDRKH interaction with MIWI results in attenuation of piRNA amplification. We find that piRNA amplification is necessary for transposon control and for sustaining piRNA levels including select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as self-serving genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
Asunto(s)
Arginina , Proteínas Argonautas , Fase Paquiteno , ARN Interferente Pequeño , Espermatogénesis , Animales , Masculino , Ratones , Arginina/metabolismo , Arginina/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Elementos Transponibles de ADN , ARN de Interacción con Piwi , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Dominio TudorRESUMEN
N-terminal arginine (NTR) methylation is a conserved feature of PIWI proteins, which are central components of the PIWI-interacting RNA (piRNA) pathway. The significance and precise function of PIWI NTR methylation in mammals remains unknown. In mice, PIWI NTRs bind Tudor domain containing proteins (TDRDs) that have essential roles in piRNA biogenesis and the formation of the chromatoid body. Using mouse MIWI (PIWIL1) as paradigm, we demonstrate that the NTRs are essential for spermatogenesis through the regulation of transposons and gene expression. Surprisingly, the loss of TDRD5 and TDRKH interaction with MIWI results in defective piRNA amplification, rather than an expected failure of piRNA biogenesis. We find that piRNA amplification is necessary for both transposon control and for sustaining levels of select, nonconserved, pachytene piRNAs that target specific mRNAs required for spermatogenesis. Our findings support the notion that the vast majority of pachytene piRNAs are dispensable, acting as autonomous genetic elements that rely for propagation on MIWI piRNA amplification. MIWI-NTRs also mediate interactions with TDRD6 that are necessary for chromatoid body compaction. Furthermore, MIWI-NTRs promote stabilization of spermiogenic transcripts that drive nuclear compaction, which is essential for sperm formation. In summary, the NTRs underpin the diversification of MIWI protein function.
RESUMEN
Transposable elements constitute nearly half of the mammalian genome and play important roles in genome evolution. While a multitude of both transcriptional and post-transcriptional mechanisms exist to silence transposable elements, control of transposition in vivo remains poorly understood. MOV10, an RNA helicase, is an inhibitor of mobilization of retrotransposons and retroviruses in cell culture assays. Here we report that MOV10 restricts LINE1 retrotransposition in mice. Although MOV10 is broadly expressed, its loss causes only incomplete penetrance of embryonic lethality, and the surviving MOV10-deficient mice are healthy and fertile. Biochemically, MOV10 forms a complex with UPF1, a key component of the nonsense-mediated mRNA decay pathway, and primarily binds to the 3' UTR of somatically expressed transcripts in testis. Consequently, loss of MOV10 results in an altered transcriptome in testis. Analyses using a LINE1 reporter transgene reveal that loss of MOV10 leads to increased LINE1 retrotransposition in somatic and reproductive tissues from both embryos and adult mice. Moreover, the degree of LINE1 retrotransposition inhibition is dependent on the Mov10 gene dosage. Furthermore, MOV10 deficiency reduces reproductive fitness over successive generations. Our findings demonstrate that MOV10 attenuates LINE1 retrotransposition in a dosage-dependent manner in mice.
Asunto(s)
Elementos Transponibles de ADN , ARN Helicasas , Animales , Masculino , Ratones , Degradación de ARNm Mediada por Codón sin Sentido , Retroelementos/genética , ARN Helicasas/genética , ARN Helicasas/metabolismoRESUMEN
Glioblastoma is the most common aggressive adult brain tumor. Numerous studies have reported results from either private institutional data or publicly available datasets. However, current public datasets are limited in terms of: a) number of subjects, b) lack of consistent acquisition protocol, c) data quality, or d) accompanying clinical, demographic, and molecular information. Toward alleviating these limitations, we contribute the "University of Pennsylvania Glioblastoma Imaging, Genomics, and Radiomics" (UPenn-GBM) dataset, which describes the currently largest publicly available comprehensive collection of 630 patients diagnosed with de novo glioblastoma. The UPenn-GBM dataset includes (a) advanced multi-parametric magnetic resonance imaging scans acquired during routine clinical practice, at the University of Pennsylvania Health System, (b) accompanying clinical, demographic, and molecular information, (d) perfusion and diffusion derivative volumes, (e) computationally-derived and manually-revised expert annotations of tumor sub-regions, as well as (f) quantitative imaging (also known as radiomic) features corresponding to each of these regions. This collection describes our contribution towards repeatable, reproducible, and comparative quantitative studies leading to new predictive, prognostic, and diagnostic assessments.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Adulto , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatología , Genómica , Glioblastoma/diagnóstico por imagen , Glioblastoma/genética , Glioblastoma/fisiopatología , Humanos , Imagen por Resonancia Magnética , PronósticoRESUMEN
CLIP-Seq (Deep Sequencing after in vivo Crosslinking and Immunoprecipitation, HITS-CLIP) has emerged as a key method for the study of RNA-binding proteins (RBPs), as it can scrutinize the RNAs bound by an RBP in vivo, with minimum manipulation of biological samples. CLIP-Seq is best used to reveal changes of the RNA cargo of an RBP and differences on binding patterns of the bound RNAs in living cells in different genetic backgrounds or after experimental treatment, rather than simply identifying RNA species. It is therefore crucial that a reference of the steady state levels of the RNAs present in the samples used for the CLIP-Seq experiment is included in the bioinformatic analysis. A simple directional RNA-Seq method was developed that uses the same oligonucleotides and the same PCR amplification steps as our CLIP-Seq method, which therefore can be analyzed using the same bioinformatic pipeline as the CLIP-Seq data. This greatly simplifies and streamlines the analysis process, and at the same time reduces the chances of protocol-specific artifacts and biases interfering with data interpretation. Some considerations on ways to integrate CLIP-Seq and RNA-Seq analyses are also provided herein.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Sitios de Unión/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoprecipitación , ARN/genética , RNA-Seq , Análisis de Secuencia de ARN/métodosRESUMEN
Direct sequencing of single, native RNA molecules through nanopores has a strong potential to transform research in all aspects of RNA biology and clinical diagnostics. The existing platform from Oxford Nanopore Technologies is unable to sequence the very 5' ends of RNAs and is limited to polyadenylated molecules. Here, we develop True End-to-end RNA Sequencing (TERA-Seq), a platform that addresses these limitations, permitting more thorough transcriptome characterization. TERA-Seq describes both poly- and non-polyadenylated RNA molecules and accurately identifies their native 5' and 3' ends by ligating uniquely designed adapters that are sequenced along with the transcript. We find that capped, full-length mRNAs in human cells show marked variation of poly(A) tail lengths at the single molecule level. We report prevalent capping downstream of canonical transcriptional start sites in otherwise fully spliced and polyadenylated molecules. We reveal RNA processing and decay at single molecule level and find that mRNAs decay cotranslationally, often from their 5' ends, while frequently retaining poly(A) tails. TERA-Seq will prove useful in many applications where true end-to-end direct sequencing of single, native RNA molecules and their isoforms is desirable.
Asunto(s)
ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Transcriptoma , Células HeLa , Humanos , Poliadenilación , Empalme del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/normasRESUMEN
We report a 57-year-old man with recurrent meningoencephalitis resulting in bouts of altered consciousness, encephalopathy, tremors, focal seizures, and paraparesis. The neurological manifestations were accompanied by fever and leukocytosis in the absence of other systemic manifestations. MRI abnormalities of the brain, brainstem, spinal cord and meninges and CSF pleocytosis and elevated protein were observed. Exhaustive studies failed to reveal an etiology. Brain biopsy revealed nodules of neutrophils and macrophages, but no vasculitis. The lesions were not vasocentric as would be expected with neuro-Behcet's disease and neuro-Sweet's disease. The disorder was responsive to high-dose corticosteroid therapy and, ultimately, to anakinra, an IL-1α and IL-1ß receptor antagonist.
Asunto(s)
Síndrome de Behçet , Meningoencefalitis , Síndrome de Sweet , Humanos , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Imagen por Resonancia Magnética , Masculino , Meningoencefalitis/tratamiento farmacológico , Persona de Mediana EdadRESUMEN
Aub guided by piRNAs ensures genome integrity by cleaving retrotransposons, and genome propagation by trapping mRNAs to form the germplasm that instructs germ cell formation. Arginines at the N-terminus of Aub (Aub-NTRs) interact with Tudor and other Tudor domain-containing proteins (TDRDs). Aub-TDRD interactions suppress active retrotransposons via piRNA amplification and form germplasm via generation of Aub-Tudor ribonucleoproteins. Here, we show that Aub-NTRs are dispensable for primary piRNA biogenesis but essential for piRNA amplification and that their symmetric dimethylation is required for germplasm formation and germ cell specification but largely redundant for piRNA amplification.
Asunto(s)
Proteínas Argonautas/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Amplificación de Genes , Células Germinativas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN Interferente Pequeño/genética , Dominio Tudor/genética , Animales , Animales Modificados Genéticamente , Arginina/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/genética , Femenino , Masculino , Ovario/metabolismo , Factores de Iniciación de Péptidos/genética , ARN Mensajero/metabolismoRESUMEN
The microRNA (miRNA) locus miR-144/451 is abundantly expressed in erythrocyte precursors, facilitating their terminal maturation and protecting against oxidant stress. However, the full repertoire of erythroid miR-144/451 target messenger RNAs (mRNAs) and associated cellular pathways is unknown. In general, the numbers of mRNAs predicted to be targeted by an miRNA vary greatly from hundreds to thousands, and are dependent on experimental approaches. To comprehensively and accurately identify erythroid miR-144/451 target mRNAs, we compared gene knockout and wild-type fetal liver erythroblasts by RNA sequencing, quantitative proteomics, and RNA immunoprecipitation of Argonaute (Ago), a component of the RNA-induced silencing complex that binds miRNAs complexed to their target mRNAs. Argonaute bound â¼1400 erythroblast mRNAs in a miR-144/451-dependent manner, accounting for one-third of all Ago-bound mRNAs. However, only â¼100 mRNAs were stabilized after miR-144/451 loss. Thus, miR-144 and miR-451 deregulate <10% of mRNAs that they bind, a characteristic that likely applies generally to other miRNAs. Using stringent selection criteria, we identified 53 novel miR-144/451 target mRNAs. One of these, Cox10, facilitates the assembly of mitochondrial electron transport complex IV. Loss of miR-144/451 caused increased Cox10 mRNA and protein, accumulation of complex IV, and increased mitochondrial membrane potential with no change in mitochondrial mass. Thus, miR-144/451 represses mitochondrial respiration during erythropoiesis by inhibiting the production of Cox10.
Asunto(s)
Transferasas Alquil y Aril/biosíntesis , Eritropoyesis/genética , Regulación de la Expresión Génica/genética , Proteínas de la Membrana/biosíntesis , MicroARNs/genética , Transferasas Alquil y Aril/genética , Animales , Proteínas de la Membrana/genética , Ratones , Ratones NoqueadosRESUMEN
Advances in RNA-sequencing methods have uncovered many aspects of RNA metabolism but are limited to surveying either the 3' or 5' terminus of RNAs, thus missing mechanistic aspects that could be revealed if both ends were captured. We developed Akron sequencing (Akron-seq), a method that captures in parallel the native 5' ends of uncapped, polyadenylated mRNAs and 3' ends of capped mRNAs from the same input RNA. Thus, Akron-seq uniquely enables assessment of full-length and truncated mRNAs at single-nucleotide resolution. Akron-seq involves RNA isolation, depletion of ribosomal and abundant small capped RNAs, and selection of capped and polyadenylated mRNAs. The endogenous ends of mRNAs are marked by adaptor ligation, followed by fragmentation, cDNA generation, PCR amplification, and deep sequencing. The step-by-step protocol we describe here is optimized for cultured human cells but can be adapted to primary cells and tissues. Akron-seq can be completed within 6 d, and sequencing and analysis can be completed within 6 d.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/genética , Análisis de Secuencia de ARN/métodos , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa , Caperuzas de ARN/genéticaRESUMEN
A crystallization-sapphire-derived-fiber (CSDF)-based Fabry-Perot interferometer (FPI) for refractive index (RI) and high-temperature measurement is proposed and demonstrated. The FPI is formed by splicing sapphire-derived fiber (SDF) to the end face of a well-cleaved single-mode fiber (SMF). CSDF is generated hundreds of micrometers away from the fusion joint resulting from arc discharge and then cuts the SDF to the edge of the CSDF. The FPI consists of two cavities, one of which is formed by CSDF, and the other is SDF, between the SMF and CSDF. The fringe contrast of the reflection spectrum varying with the RI changes of the external environment is used for RI sensing, while the wavelength shifting is for the ambient temperature sensing. In the experiment, the refractive index and temperature sensitivities are about 233.8 dB/RIU in the RI range of 1.333-1.363 and 13.571 pm/°C in the temperature range of 20°C-1000°C.
RESUMEN
In this issue of Molecular Cell, Gainetdinov et al. (2018) show that PIWI proteins direct both piRNA biogenesis and piRNA function in most animals.
Asunto(s)
Proteínas , Animales , ARN Interferente PequeñoRESUMEN
mRNAs transmit the genetic information that dictates protein production and are a nexus for numerous pathways that regulate gene expression. The prevailing view of canonical mRNA decay is that it is mediated by deadenylation and decapping followed by exonucleolysis from the 3' and 5' ends. By developing Akron-seq, a novel approach that captures the native 3' and 5' ends of capped and polyadenylated RNAs, respectively, we show that canonical human mRNAs are subject to repeated cotranslational and ribosome-phased endonucleolytic cuts at the exit site of the mRNA ribosome channel, in a process that we term ribothrypsis. We uncovered RNA G quadruplexes among likely ribothrypsis triggers and show that ribothrypsis is a conserved process. Strikingly, we found that mRNA fragments are abundant in living cells and thus have important implications for the interpretation of experiments, such as RNA-seq, that rely on the assumption that mRNAs exist largely as full-length molecules in vivo.
Asunto(s)
G-Cuádruplex , Estabilidad del ARN/genética , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Exorribonucleasas/metabolismo , Biblioteca de Genes , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Sistemas de Lectura Abierta , Caperuzas de ARN/metabolismo , Análisis de Secuencia de ARNRESUMEN
HITS-CLIP (High-Throughput Sequencing after in vivo Crosslinking and Immunoprecipitation, CLIP-Seq) libraries contain fragments of the RNA sequences bound in vivo by an RNA binding protein (RBP). Such fragments, especially if they represent RNA duplexes bound in vivo by the RBP, can occasionally be ligated together to form chimeric CLIP tags. Chimeric CLIP tags from Argonaute CLIP libraries can provide the exact base pairing profiles of small RNAs with their target RNA sequences, thus solving a critical problem in the field of post-transcriptional regulation. We recently reported an analysis of chimeric reads from the Drosophila Piwi protein Aubergine, which revealed a novel mechanism for mRNA entrapment within germ RNP granules. We term this novel approach chimeric CLIP (cCLIP) and present here the main steps that a researcher can take after the acquisition of the deep sequencing data, for the identification of candidate chimeric reads in Piwi CLIP libraries. Extending the scope beyond small-RNA binding proteins, we believe that cCLIP can be utilized to elucidate the in vivo functions of RNA-binding proteins in general, and especially those that modulate RNA secondary structures. We, therefore, also describe aspects of the generalized chimeric read identification problem, which can find use in the analysis of the CLIP libraries of any RNA-binding protein.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación , Análisis de Secuencia de ARN , Proteínas Argonautas , Emparejamiento Base , Biología Computacional/métodos , Proteínas de Drosophila , Perfilación de la Expresión Génica , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunoprecipitación/métodos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Programas InformáticosRESUMEN
The GD2 ganglioside, which is abundant on the surface of neuroblastoma cells, is targeted by an FDA-approved therapeutic monoclonal antibody and is an attractive tumor-associated antigen for cellular immunotherapy. Chimeric antigen receptor (CAR)-modified T cells can have potent antitumor activity in B-cell malignancies, and trials to harness this cytolytic activity toward GD2 in neuroblastoma are under way. In an effort to enhance the antitumor activity of CAR T cells that target GD2, we generated variant CAR constructs predicted to improve the stability and the affinity of the GD2-binding, 14G2a-based, single-chain variable fragment (scFv) of the CAR and compared their properties in vivo We included the E101K mutation of GD2 scFv (GD2-E101K) that has enhanced antitumor activity against a GD2+ human neuroblastoma xenograft in vivo However, this enhanced antitumor efficacy in vivo was concomitantly associated with lethal central nervous system (CNS) toxicity comprised of extensive CAR T-cell infiltration and proliferation within the brain and neuronal destruction. The encephalitis was localized to the cerebellum and basal regions of the brain that display low amounts of GD2. Our results highlight the challenges associated with target antigens that exhibit shared expression on critical normal tissues. Despite the success of GD2-specific antibody therapies in the treatment of neuroblastoma, the fatal neurotoxicity of GD2-specific CAR T-cell therapy observed in our studies suggests that GD2 may be a difficult target antigen for CAR T-cell therapy without additional strategies that can control CAR T-cell function within the CNS. Cancer Immunol Res; 6(1); 36-46. ©2017 AACR.
Asunto(s)
Encefalitis/etiología , Gangliósidos/inmunología , Inmunoterapia Adoptiva , Neuroblastoma/complicaciones , Neuroblastoma/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Animales , Complejo CD3/genética , Línea Celular Tumoral , Proliferación Celular , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Encefalitis/diagnóstico , Gangliósidos/metabolismo , Orden Génico , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Ratones , Neuroblastoma/terapia , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PIWI family proteins bind to small RNAs known as PIWI-interacting RNAs (piRNAs) and play essential roles in the germline by silencing transposons and by promoting germ cell specification and function. Here we report that the widely used Kc167 cell line, derived from Drosophila melanogaster embryos, expresses piRNAs that are loaded to Aub and Piwi. Kc167 piRNAs are produced by a canonical, primary piRNA biogenesis pathway, from phased processing of precursor transcripts by the Zuc endonuclease, Armi helicase, and dGasz mitochondrial scaffold protein. Kc167 piRNAs derive from cytoplasmic transcripts, notably tRNAs and mRNAs, and their abundance correlates with that of parent transcripts. The expression of Aub is robust in Kc167, that of Piwi is modest, while Ago3 is undetectable, explaining the lack of transposon-related piRNA amplification by the Aub-Ago3, ping-pong mechanism. We propose that the default state of the primary piRNA biogenesis machinery is random transcript sampling to allow generation of piRNAs from any transcript, including newly acquired retrotransposons. This state is unmasked in Kc167, likely because they do not express piRNA cluster transcripts in sufficient amounts and do not amplify transposon piRNAs. We use Kc167 to characterize an inactive isoform of Aub protein. Since most Kc167 piRNAs are genic, they can be mapped uniquely to the genome, facilitating computational analyses. Furthermore, because Kc167 is a widely used and well-characterized cell line that is easily amenable to experimental manipulations, we expect that it will serve as an excellent system to study piRNA biogenesis and piRNA-related factors.
Asunto(s)
Drosophila melanogaster/genética , ARN Interferente Pequeño/genética , Animales , Proteínas Argonautas/metabolismo , Línea Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Genoma de los Insectos , Factores de Iniciación de Péptidos/metabolismo , ARN Mensajero/genética , ARN de Transferencia/genética , Transducción de SeñalRESUMEN
The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) have a central role in genomic stability, which is inextricably linked to germ-cell formation, by forming Piwi ribonucleoproteins (piRNPs) that silence transposable elements. In Drosophila melanogaster and other animals, primordial germ-cell specification in the developing embryo is driven by maternal messenger RNAs and proteins that assemble into specialized messenger ribonucleoproteins (mRNPs) localized in the germ (pole) plasm at the posterior of the oocyte. Maternal piRNPs, especially those loaded on the Piwi protein Aubergine (Aub), are transmitted to the germ plasm to initiate transposon silencing in the offspring germ line. The transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process; mRNAs necessary for primordial germ-cell formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, the molecular identity of which remains unknown. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm, and interactions between Aub and Tudor are essential for the formation of germ granules. Here we show that Aub-loaded piRNAs use partial base-pairing characteristics of Argonaute RNPs to bind mRNAs randomly in Drosophila, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Notably, germ plasm mRNAs in drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus, complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. Our findings reveal an unexpected function for piRNP complexes in mRNA trapping that may be generally relevant to the function of animal germ granules.
Asunto(s)
Citoplasma/genética , Citoplasma/metabolismo , Drosophila melanogaster/genética , Oocitos/citología , Transporte de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Animales , Proteínas Argonautas/metabolismo , Emparejamiento Base , Sitios de Unión , Elementos Transponibles de ADN/genética , Difusión , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Femenino , Masculino , Proteínas de Transporte de Membrana/metabolismo , Oocitos/metabolismo , Oogénesis , Factores de Iniciación de Péptidos/metabolismo , Interferencia de ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Ribonucleoproteínas/metabolismo , Transcriptoma/genéticaRESUMEN
Immunoprecipitation of RNA binding proteins (RBPs) after in vivo crosslinking, coupled with sequencing of associated RNA footprints (HITS-CLIP, CLIP-seq), is a method of choice for the identification of RNA targets and binding sites for RBPs. Compared with RNA-seq, CLIP-seq analysis is widely diverse and depending on the RBPs that are analyzed, the approaches vary significantly, necessitating the development of flexible and efficient informatics tools. In this study, we present CLIPSeqTools, a novel, highly flexible computational suite that can perform analysis from raw sequencing data with minimal user input. It contains a wide array of tools to provide an in-depth view of CLIP-seq data sets. It supports extensive customization and promotes improvization, a critical virtue, since CLIP-seq analysis is rarely well defined a priori. To highlight CLIPSeqTools capabilities, we used the suite to analyze Ago-miRNA HITS-CLIP data sets that we prepared from human brains.