RESUMO
Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3' splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the beta-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between IIIa and beta-globin in order to identify the minimal IIIa sequence element conferring enhanced splicing in Ad-NE. Using this approach, we show that the IIIa branch site/pyrimidine tract functions as a Janus element: it blocks splicing in HeLa-NE and functions as a splicing enhancer in Ad-NE. Therefore, we named this sequence the IIIa virus infection-dependent splicing enhancer (3VDE). This element is essential for regulated IIIa pre-mRNA splicing in Ad-NE and sufficient to confer an enhanced splicing phenotype to the beta-globin pre-mRNA in Ad-NE. We further show that the increase in IIIa splicing observed in Ad-NE is not accompanied by a similar increase in U2AF binding to the IIIa pyrimidine tract. This finding suggests that splicing activation by the 3VDE may operate without efficient U2AF interaction with the pre-mRNA. Importantly, this report represents the first description of a splicing enhancer that has evolved to function selectively in the context of a virus infection, a finding that adds a new level at which viruses may subvert the host cell RNA biosynthetic machinery to facilitate their own replication.
Assuntos
Adenoviridae/genética , Regulação Viral da Expressão Gênica/genética , Precursores de RNA/metabolismo , Splicing de RNA , Globinas/genética , Células HeLa , Humanos , Mutação , Proteínas Nucleares , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Ribonuclease H/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas/genética , Fator de Processamento U2AFRESUMO
Mammalian proteins expressed in Escherichia coli are used in a variety of applications. A major drawback in producing eukaryotic proteins in E.coli is that the bacteria lack most eukaryotic post-translational modification systems, including serine/threonine protein kinase(s). Here we show that a eukaryotic protein can be phosphorylated in E.coli by simultaneous expression of a mammalian protein kinase and its substrate. We show that in bacteria expressing SRPK1, ASF/SF2 becomes phosphorylated to a degree resembling native ASF/SF2 present in interphase HeLa cell nuclei. The E.coli phosphorylated ASF/SF2 is functional in splicing and, contrary to the unphosphorylated protein, soluble under native conditions.
Assuntos
Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Splicing de RNA , Clonagem Molecular , Escherichia coli , Células HeLa , Humanos , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Processamento de Serina-Arginina , SolubilidadeRESUMO
Splicing enhancers have previously been shown to promote processing of introns containing weak splicing signals. Here, we extend these studies by showing that also 'strong' constitutively active introns are absolutely dependent on a downstream splicing enhancer for activity in vitro. SR protein binding to exonic enhancer elements or U1 snRNP binding to a downstream 5' splice site serve redundant functions as activators of splicing. We further show that a 5' splice site is most effective as an enhancer of splicing. Thus, a 5' splice site is functional in S100 extracts, under conditions where a SR enhancer is nonfunctional. Also, splice site pairing occurs efficiently in the absence of exonic SR enhancers, emphasizing the significance of a downstream 5' splice site as the enhancer element in vertebrate splicing.