RESUMO
DNA mismatch recognition is performed in eukaryotes by two heterodimers known as MutSalpha (Msh2/Msh6) and MutSbeta (Msh2/Msh3) that must reside in the nucleus to function. Two putative Msh2 nuclear localization sequences (NLS) were characterized by fusion to green fluorescent protein (GFP) and site-directed mutagenesis in the context of Msh2. One NLS functioned in GFP targeting assays and both acted redundantly within Msh2. We examined nuclear localization of each of the MutS monomers in the presence and absence of their partners. Msh2 translocated to the nucleus in cells lacking Msh3 and Msh6; however, cells lacking Msh6 showed significantly decreased levels of nuclear Msh2. Furthermore, the overall protein levels of Msh2 were significantly diminished in the absence of Msh6, particularly if Msh2 lacked a functional NLS. Msh3 localized in the absence of Msh2, but Msh6 localization depended on Msh2 expressing functional NLSs. Overall, the nuclear levels of Msh2 and Msh6 decline when the other partner is absent. The data suggest a stabilization mechanism to prevent free monomer accumulation in the cytoplasm.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/genética , Citoplasma/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Proteína 2 Homóloga a MutS/genética , Mutagênese Sítio-Dirigida , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The pathway of gene expression in higher eukaryotes involves a highly complex network of physical and functional interactions among the different machines involved in each step of the pathway. Here we established an efficient in vitro system to determine how RNA polymerase II (RNAP II) transcription is functionally coupled to pre-mRNA splicing. Strikingly, our data show that nascent pre-messenger RNA (pre-mRNA) synthesized by RNAP II is immediately and quantitatively directed into the spliceosome assembly pathway. In contrast, nascent pre-mRNA synthesized by T7 RNA polymerase is quantitatively assembled into the nonspecific H complex, which consists of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins and is inhibitory for spliceosome assembly. Consequently, RNAP II transcription results in a dramatic increase in both the kinetics of splicing and overall yield of spliced mRNA relative to that observed for T7 transcription. We conclude that RNAP II mediates the functional coupling of transcription to splicing by directing the nascent pre-mRNA into spliceosome assembly, thereby bypassing interaction of the pre-mRNA with the inhibitory hnRNP proteins.
