RESUMO
A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6ΔΔ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin architectural proteins Nhp6A/Nhp6B, accumulate intron-containing pre-mRNA at the restrictive temperature. In addition, we demonstrate that rsc8-ts16 nhp6ΔΔ cells contain low levels of U6 snRNA and U4/U6 di-snRNA that is further exacerbated after two hours growth at the restrictive temperature. This change in U6 snRNA and U4/U6 di-snRNA levels in rsc8-ts16 nhp6ΔΔ cells is indicative of splicing deficient conditions. We identify MRN1 (multi-copy suppressor of rsc nhp6ΔΔ) as a growth suppressor of rsc nhp6ΔΔ synthetic sickness. Mrn1 is an RNA binding protein that localizes both to the nucleus and cytoplasm. Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309Δ, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas HMGN/genética , Proteínas HMGN/metabolismo , Immunoblotting , Microscopia de Fluorescência , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Homologous recombination is an important pathway for the repair of DNA double-strand breaks (DSBs). In the yeast Saccharomyces cerevisiae, Rad52 is a central recombination protein, whereas its paralogue, Rad59, plays a more subtle role in homologous recombination. Both proteins can mediate annealing of complementary single-stranded DNA in vitro, but only Rad52 interacts with replication protein A and the Rad51 recombinase. We have studied the functional overlap between Rad52 and Rad59 in living cells using chimeras of the two proteins and site-directed mutagenesis. We find that Rad52 and Rad59 have both overlapping as well as separate functions in DSB repair. Importantly, the N-terminus of Rad52 possesses functions not supplied by Rad59, which may account for its central role in homologous recombination.
Assuntos
Reparo do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
We have identified carbon catabolite repression (CCR) as a regulator of amino acid permeases in Saccharomyces cerevisiae, elucidated the permeases regulated by CCR, and identified the mechanisms involved in amino acid permease regulation by CCR. Transport of l-arginine and l-leucine was increased by approximately 10-25-fold in yeast grown in carbon sources alternate to glucose, indicating regulation by CCR. In wild type yeast the uptake (pmol/10(6) cells/h), in glucose versus galactose medium, of l-[(14)C]arginine was (0.24 +/- 0.04 versus 6.11 +/- 0.42) and l-[(14)C]leucine was (0.30 +/- 0.02 versus 3.60 +/- 0.50). The increase in amino acid uptake was maintained when galactose was replaced with glycerol. Deletion of gap1Delta and agp1Delta from the wild type strain did not alter CCR induced increase in l-leucine uptake; however, deletion of further amino acid permeases reduced the increase in l-leucine uptake in the following manner: 36% (gnp1Delta), 62% (bap2Delta), 83% (Delta(bap2-tat1)). Direct immunofluorescence showed large increases in the expression of Gnp1 and Bap2 proteins when grown in galactose compared with glucose medium. By extending the functional genomic approach to include major nutritional transducers of CCR in yeast, we concluded that SNF/MIG, GCN, or PSK pathways were not involved in the regulation of amino acid permeases by CCR. Strikingly, the deletion of TOR1, which regulates cellular response to changes in nitrogen availability, from the wild type strain abolished the CCR-induced amino acid uptake. Our results provide novel insights into the regulation of yeast amino acid permeases and signaling mechanisms involved in this regulation.