RESUMEN
SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript. The first half acts as dominant-negative isoform suppressing poison cassette exon inclusion and instead promoting the retention of flanking introns containing repeated SRSF7 binding sites. Massive SRSF7 binding to these sites and its oligomerization promote the assembly of large nuclear bodies, which sequester SRSF7 transcripts at their transcription site, preventing their export and restoring normal SRSF7 protein levels. We further show that hundreds of human and mouse NMD targets, especially RNA-binding proteins, encode potential Split-ORFs, some of which are expressed under specific cellular conditions.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Neoplasias/genética , Sistemas de Lectura Abierta , Precursores del ARN/genética , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Exones , Homeostasis/genética , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Proteínas de Neoplasias/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Unión Proteica , Biosíntesis de Proteínas , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/clasificación , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Transcripción GenéticaRESUMEN
SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.