RESUMEN
Rapamycin is a naturally occurring macrolide whose target is at the core of nutrient and stress regulation in a wide range of species. Despite well-established roles as an inhibitor of cap-dependent mRNA translation, relatively little is known about its effects on other modes of RNA processing. Here, we characterize the landscape of rapamycin-induced post-transcriptional gene regulation. Transcriptome analysis of rapamycin-treated cells reveals genome-wide changes in alternative mRNA splicing and pronounced changes in NMD-sensitive isoforms. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD of certain transcripts. Rapamycin-treatment significantly reduces the levels of both endogenous and exogenous Premature Termination Codon (PTC)-containing mRNA isoforms and its effects are dose-, UPF1- and 4EBP-dependent. The PTC-containing SRSF6 transcript exhibits a shorter half-life upon rapamycin-treatment as compared to the non-PTC isoform. Rapamycin-treatment also causes depletion of PTC-containing mRNA isoforms from polyribosomes, underscoring the functional relationship between translation and NMD. Enhanced NMD activity also correlates with an enrichment of the nuclear Cap Binding Complex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin modulates global RNA homeostasis by NMD.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido/efectos de los fármacos , Sirolimus/farmacología , Empalme Alternativo/efectos de los fármacos , Codón sin Sentido , Factores Eucarióticos de Iniciación/fisiología , Células HEK293 , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Polirribosomas/metabolismo , ARN Helicasas , Isoformas de ARN/metabolismo , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , Transactivadores/fisiologíaRESUMEN
Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3. These transcripts encode proteins enriched for functions such as cell migration, proliferation, and adhesion. Loss of IGF2BP3 reduced PDAC cell invasiveness and remodeled focal adhesion junctions. Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) revealed significant overlap of IGF2BP3 and microRNA (miRNA) binding sites. IGF2BP3 promotes association of the RNA-induced silencing complex (RISC) with specific transcripts. Our results show that IGF2BP3 influences a malignancy-associated RNA regulon by modulating miRNA-mRNA interactions.
