RESUMO
Cyclin D1 expression represents one of the key mitogen-regulated events during the G(1) phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP-associated RNA-processing complex, is coordinately recruited to both the 3' end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer.
Assuntos
Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA Neoplásico/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Ciclina D1/biossíntese , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AF , Transcrição Gênica , TransfecçãoRESUMO
We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.
