RESUMO
Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.
Assuntos
Encéfalo , RNA Circular , Transmissão Sináptica , RNA Circular/genética , Animais , Camundongos , Encéfalo/metabolismo , Encéfalo/fisiologia , Camundongos Knockout , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
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
Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.
Assuntos
Processamento Alternativo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Amanitinas/farmacologia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Proteínas de Drosophila/genética , Drosophila melanogaster , Éxons , Fibronectinas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Modelos Biológicos , Modelos Genéticos , Plasmídeos/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Isoformas de Proteínas , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , 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
A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism. The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family. In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs. TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark. Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs. In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene. When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2. This inhibition was similar to that observed with human SRPK1. This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism. The implications in the origins and control of splicing are discussed.
Assuntos
Genes de Protozoários , Proteínas Serina-Treonina Quinases/genética , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Clonagem Molecular , Globinas/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Schizosaccharomyces/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Trypanosoma cruzi/genéticaRESUMO
A novel serine-arginine-rich protein designated TcSR was identified in Trypanosoma cruzi. The deduced amino acid sequence reveals that TcSR is a member of the SR protein family of splicing factors that contains two RNA-binding domains at the N-terminal side and several serine-arginine repeats at the COOH-terminus. Over expression of either TcSR or the human SR-protein associated splicing factor/splicing factor 2 (ASF/SF2) in wild-type Schizosaccharomyces pombe, provoked an elongated phenotype similar to that of fission yeast over expressing the SR-containing splicing factor Prp2, a U2AF(65) orthologue. When a double mutant strain lacking two SR protein-specific protein kinases was used, expression of TcSR or human SR ASF/SF2 splicing factor reverted the mutant to a wild-type phenotype. Transient expression of TcSR in HeLa cells stimulated the inclusion of the EDI exon of human fibronectin in an in vivo functional alternative cis-splicing assay. Inclusion was dependent on a splicing enhancer sequence present in the EDI exon. In addition, TcSR and peptides carrying TcSR-RS domain sequences were phosphorylated by a human SR protein kinase. These results indicate that TcSR is a member of the SR splicing network and that some components common to the trans- and cis-splicing machineries evolved from the early origins of the eukaryotic lineage.
Assuntos
Arginina/análise , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Splicing de RNA , Serina/análise , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Evolução Molecular , Células HeLa , Humanos , Proteínas de Protozoários/genética , Schizosaccharomyces/genética , Alinhamento de Sequência , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/fisiologiaRESUMO
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.
Assuntos
Processamento Alternativo , Fibronectinas/genética , Vírus 40 dos Símios/genética , Transativadores/metabolismo , Transcrição Gênica , Animais , Antígenos Transformantes de Poliomavirus/genética , Células COS , Chlorocebus aethiops , Elementos Facilitadores Genéticos , Éxons , Regulação Viral da Expressão Gênica/fisiologia , RNA Polimerase II/genética , Origem de Replicação , TransfecçãoRESUMO
Promoters and enhancers are cis-acting elements that control gene transcription via complex networks of protein-DNA and protein-protein interactions. Whereas promoters deal with putting in place the RNA polymerase, both enhancers and promoters can control transcriptional initiation and elongation. We have previously shown that promoter structure modulates alternative splicing, strengthening the concept of a physical and functional coupling between transcription and splicing. Here we report that the promoter effect is due to the control of RNA pol II elongation. We found that the simian virus 40 (SV40) transcriptional enhancer, inserted in fibronectin (FN) minigene constructs transfected into mammalian cells, controls alternative splicing by inhibiting inclusion of the FN extra domain I (EDI) exon into mature mRNA. Deletion analysis of enhancer subdomains and competitions in vivo with excess of specific enhancer DNA subfragments demonstrate that the "minimal" enhancer, consisting of two 72-bp repeats, is responsible for the splicing effect. The 72-bp repeat region has been reported to promote RNA pol II elongation. When transcription is driven by the alpha-globin promoter linked to the SV40 enhancer, basal EDI inclusion and activation by the SR (Ser-Arg-rich) protein SF2/ASF are much lower than with other promoters. Deletion of only one of the two 72-bp repeats not only provokes higher EDI inclusion levels but allows responsiveness to SF2/ASF. These effects are the consequence of a decrease in RNA pol II elongation evidenced both by an increase in the proportions of shorter proximal over full length transcripts and by higher pol II densities upstream of the alternative exon detected by chromatin immunoprecipitation.