RESUMO
Alternative splicing plays a key role in generating protein diversity. Transfections with minigenes revealed coordination between two distant, alternatively spliced exons in the same gene. Mutations that either inhibit or stimulate inclusion of the upstream alternative exon deeply affect inclusion of the downstream one. However, similar mutations at the downstream alternative exon have little effect on the upstream one. This polar effect is promoter specific and is enhanced by inhibition of transcriptional elongation. Consistently, cells from mutant mice with either constitutive or null inclusion of a fibronectin alternative exon revealed coordination with a second alternative splicing region, located far downstream. Using allele-specific RT-PCR, we demonstrate that this coordination occurs in cis and is also affected by transcriptional elongation rates. Bioinformatics supports the generality of these findings, indicating that 25% of human genes contain multiple alternative splicing regions and identifying several genes with nonrandom distribution of mRNA isoforms at two alternative regions.
Assuntos
Processamento Alternativo , Genes/genética , Alelos , alfa-Globulinas/genética , Animais , Antígenos Virais de Tumores/genética , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Biologia Computacional , Proteínas de Ligação a DNA/genética , Diclororribofuranosilbenzimidazol/farmacologia , Éxons/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Humanos , Camundongos , Camundongos Knockout , Modelos Genéticos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina , Fatores de Transcrição/genética , TransfecçãoRESUMO
Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.
Assuntos
Splicing de RNA , Transcrição Gênica , Processamento Alternativo , Animais , Humanos , Modelos Biológicos , Subunidades Proteicas , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Spliceossomos/metabolismo , Transativadores/metabolismo , Trans-SplicingRESUMO
Transcription and pre-mRNA splicing are coordinated temporally and spatially, and both processes can influence each other. In particular, control of transcriptional elongation by RNA polymerase II has proved to be important for alternative splicing regulation. In this report we demonstrate that the efficiency of exon recognition by the splicing machinery is crucial for the elongation control. Alternative splicing of the fibronectin extra domain I (EDI) is because the polypyrimidine tract of its 3'-splice site occurs suboptimal. By mutating the polypyrimidine tract of EDI in two different positions, individually or in combination, and by disrupting its exonic splicing silencer, we managed to generate minigenes with increasing degrees of exon recognition. Improvement of exon recognition is evidenced by independence from the splicing regulator SF2/ASF for inclusion. The mutated minigenes were used to transfect human cells in culture and study the responsiveness of EDI alternative splicing to activation or inhibition of pol II elongation. Our results revealed that responsiveness of exon skipping to elongation is inversely proportional to 3'-splice site strength, which means that the better the alternative exon is recognized by the splicing machinery, the less its degree of inclusion is affected by transcriptional elongation.
Assuntos
Processamento Alternativo , RNA Polimerase II/química , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Éxons , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Humanos , Modelos Biológicos , Mutação , Fosforilação , Plasmídeos/metabolismo , Mutação Puntual , Polimorfismo Genético , Estrutura Terciária de Proteína , Pirimidinas/química , RNA Polimerase II/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
The realization that the mammalian proteomic complexity is achieved with a limited number of genes demands a better understanding of alternative splicing regulation. Promoter control of alternative splicing was originally described by our group in studies performed on the fibronectin gene. Recently, other labs extended our findings to the cystic fibrosis, CD44 and CGRP genes strongly supporting a coupling between transcription and pre-mRNA splicing. A possible mechanism that would fit in these results is that the promoter itself is responsible for recruiting splicing factors, such as SR proteins, to the site of transcription, possibly through transcription factors that bind the promoter or the transcriptional enhancers. An alternative model, discussed more extensively in this review, involves modulation of RNA pol II (pol II) elongation rate. The model is supported by findings that cis- and trans- acting factors that modulate pol II elongation on a particular template also provoke changes in the alternative splicing balance of the encoded mRNAs.
Assuntos
Processamento Alternativo , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Animais , Modelos Genéticos , Proteínas de Ligação a RNA/metabolismo , Transcrição GênicaRESUMO
Promoter and enhancer elements can influence alternative splicing, but the basis for this phenomenon is not well understood. Here we investigated how different transcriptional activators affect the decision between inclusion and exclusion (skipping) of the fibronectin EDI exon. A mutant of the acidic VP16 activation domain called SW6 that preferentially inhibits polymerase II (pol II) elongation caused a reduction in EDI exon skipping. Exon skipping was fully restored in the presence of the SW6 mutant by either the SV40 enhancer in cis or the human immunodeficiency virus (HIV) Tat in trans, both of which specifically stimulate pol II elongation. HIV Tat also cooperated with the Sp1 and CTF activation domains to enhance transcript elongation and EDI skipping. The extent of exon skipping correlated with the efficiency with which pol II transcripts reach the 3' end of the gene but not with the overall fold increase in transcript levels caused by different activators. The ability of activators to enhance elongation by RNA polymerase II therefore correlates with their ability to enhance exon skipping. Consistent with this observation, the elongation inhibitor dichlororibofuranosylbenzimidazole (DRB) enhanced EDI inclusion. Conversely, the histone deacetylase inhibitor trichostatin A that is thought to stimulate elongation caused a modest inhibition of EDI inclusion. Together our results support a kinetic coupling model in which the rate of transcript elongation determines the outcome of two competing splicing reactions that occur co-transcriptionally. Rapid, highly processive transcription favors EDI exon skipping, whereas slower, less processive transcription favors inclusion.