RESUMO
At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes.
Assuntos
DNA Fúngico/metabolismo , Processamento de Terminações 3' de RNA , RNA Polimerase II/metabolismo , Precursores de RNA/biossíntese , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Modelos Genéticos , Ligação Proteica , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Colonization of genomes by a new selfish genetic element is detrimental to the host species and must lead to an efficient, repressive response. In vertebrates as well as in Drosophila, piRNAs repress transposons in the germ line, whereas endogenous siRNAs take on this role in somatic cells. We show that their biogenesis depends on a new isoform of the Drosophila TRBP homologue loquacious, which arises by alternative polyadenylation and is distinct from the one that functions during the biogenesis of miRNAs. For endo-siRNAs and piRNAs, it is unclear how an efficient response can be initiated de novo. Our experiments establish that the endo-siRNA pathway will target artificially introduced sequences without the need for a pre-existing template in the genome. This response is also triggered in transiently transfected cells, thus genomic integration is not essential. Deep sequencing showed that corresponding endo-siRNAs are generated throughout the sequence, but preferentially from transcribed regions. One strand of the dsRNA precursor can come from spliced mRNA, whereas the opposite strand derives from independent transcripts in antisense orientation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Splicing de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , Transfecção , TransgenesRESUMO
Development, growth and adult survival are coordinated with available metabolic resources, ascertaining that the organism responds appropriately to environmental conditions. MicroRNAs are short (21-23 nt) regulatory RNAs that confer specificity on the RNA-induced silencing complex (RISC) to inhibit a given set of mRNA targets. We profiled changes in miRNA expression during adult life in Drosophila melanogaster and determined that miR-277 is downregulated during adult life. Molecular analysis revealed that this miRNA controls branched-chain amino acid (BCAA) catabolism and as a result it can modulate the activity of the TOR kinase, a central growth regulator, in cultured cells. Metabolite analysis in cultured cells as well as flies suggests that the mechanistic basis may be an accumulation of branched-chain α-keto-acids (BCKA), rather than BCAAs, thus avoiding potentially detrimental consequences of increased branched chain amino acid levels on e.g., translational fidelity. Constitutive miR-277 expression shortens lifespan and is synthetically lethal with reduced insulin signaling, indicating that metabolic control underlies this phenotype. Transgenic inhibition with a miRNA sponge construct also shortens lifespan, in particular on protein-rich food. Thus, optimal metabolic adaptation appears to require tuning of cellular BCAA catabolism by miR-277.