RESUMEN
BACKGROUND: The exon junction complex (EJC) is involved in most steps of the mRNA life cycle, ranging from splicing to nonsense-mediated mRNA decay (NMD). It is assembled by the splicing machinery onto mRNA in a sequence-independent manner. A fundamental open question is whether the EJC is deposited onto all exonâexon junctions or only on a subset of them. Several previous studies have made observations supportive of the latter, yet these have been limited by methodological constraints. RESULTS: In this study, we sought to overcome these limitations via the integration of two different approaches for transcriptome-wide mapping of EJCs. Our results revealed that nearly all, if not all, internal exons consistently harbor an EJC in Drosophila, demonstrating that EJC presence is an inherent consequence of the splicing reaction. Furthermore, our study underscores the limitations of eCLIP methods in fully elucidating the landscape of RBP binding sites. Our findings highlight how highly specific (low false positive) methodologies can lead to erroneous interpretations due to partial sensitivity (high false negatives). CONCLUSIONS: This study contributes to our understanding of EJC deposition and its association with pre-mRNA splicing. The universal presence of EJC on internal exons underscores its significance in ensuring proper mRNA processing. Additionally, our observations highlight the need to consider both specificity and sensitivity in RBP mapping methodologies.
Asunto(s)
Proteínas de Unión al ARN , Ribonucleoproteínas , Animales , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Drosophila/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Exones , Sitios de UniónRESUMEN
CLIP-seq methods provide transcriptome-wide snapshots of RNA-protein interactions in live cells. Reverse transcriptases stopping at cross-linked nucleotides sign for RNA-protein binding sites. Reading through cross-linked positions results in false binding site assignments. In the 'monitored enhanced CLIP' (meCLIP) method, a barcoded biotinylated linker is ligated at the 5' end of cross-linked RNA fragments to purify RNA prior to the reverse transcription. cDNAs keeping the barcode sequence correspond to reverse transcription read-throughs. Read through occurs in unpredictable proportions, representing up to one fourth of total reads. Filtering out those reads strongly improves reliability and precision in protein binding site assignment.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Inmunoprecipitación/métodos , Proteínas/metabolismo , ARN/metabolismo , Sitios de Unión , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Complementario , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Humanos , Oligonucleótidos/química , Oligonucleótidos/genética , Proteínas/química , Proteínas/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Transcripción Reversa , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle. An imbalance in EJC dosage alters mouse neural stem cell (mNSC) division and is linked to human neurodevelopmental disorders. In quiescent mNSC and immortalized human retinal pigment epithelial (RPE1) cells, centrioles form a basal body for ciliogenesis. Here, we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD2. In contrast to BICD2, the localization of NIN transcripts is EJC-dependent. NIN mRNA encodes a core component of centrosomes required for microtubule nucleation and anchoring. We find that EJC down-regulation impairs both pericentriolar material organization and ciliogenesis. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development.