RESUMO
We have characterized two Saccharomyces cerevisiae proteins, Sro9p and Slf1p, which contain a highly conserved motif found in all known La proteins. Originally described as an autoantigen in patients with rheumatic disease, the La protein binds to newly synthesized RNA polymerase III transcripts. In yeast, the La protein homologue Lhp1p is required for the normal pathway of tRNA maturation and also stabilizes newly synthesized U6 RNA. We show that deletions in both SRO9 and SLF1 are not synthetically lethal with a deletion in LHP1, indicating that the three proteins do not function in a single essential process. Indirect immunofluorescence microscopy reveals that although Lhp1p is primarily localized to the nucleus, Sro9p is cytoplasmic. We demonstrate that Sro9p and Slf1p are RNA-binding proteins that associate preferentially with translating ribosomes. Consistent with a role in translation, strains lacking either Sro9p or Slf1p are less sensitive than wild-type strains to certain protein synthesis inhibitors. Thus, Sro9p and Slf1p define a new and possibly evolutionarily conserved class of La motif-containing proteins that may function in the cytoplasm to modulate mRNA translation.
Assuntos
Autoantígenos/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Polirribossomos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Autoantígenos/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Filogenia , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Alinhamento de Sequência , Antígeno SS-BRESUMO
Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells and is associated with most human cancers. In all eukaryotes examined, telomerase has an RNA subunit, a conserved reverse transcriptase subunit and additional proteins, but little is known about the assembly of these components. Here we show that the Saccharomyces cerevisiae telomerase RNA has a 5'-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing. Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex. These data support a model in which telomerase RNA is transcribed by RNA polymerase II and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits. We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication.
Assuntos
RNA Fúngico/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Telomerase/metabolismo , Autoantígenos , Genes Fúngicos , Guanosina/análogos & derivados , Guanosina/metabolismo , Capuzes de RNA , Splicing de RNA , RNA Fúngico/química , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genética , Saccharomyces cerevisiae/genética , Telomerase/biossíntese , Telomerase/química , Telomerase/genética , Telômero , Proteínas Centrais de snRNPRESUMO
Cell duplication is characteristic of life. The coordination of cell growth with cell duplication and, specifically, the ordered steps necessary for this process are termed the cell cycle. Central to this process is the faithful replication and segregation of the chromosomes. The cycle consists of four phases: G1, where the decision to enter the cell cycle, which is known as Start, is made; S phase, during which the DNA is replicated; G2, during which controls assuring the completion of S phase operate; and M, or the mitotic phase, which is characterized by chromosome segregation, nuclear division, and cytokinesis. The budding yeast Saccharomyces cerevisiae has been developed into a model genetic system for the study of the cell division cycle (Hartwell et al. ["73] Genetics, 74:267-286). Here I review the basic processes by which chromosomes are segregated, with an emphasis on the physical structures fundamental to this process.
Assuntos
Mitose , Saccharomyces cerevisiae/citologia , Fuso Acromático , Animais , Microtúbulos , Tubulina (Proteína)RESUMO
A Saccharomyces cerevisiae gamma-tubulin-related gene, TUB4, has been characterized. The predicted amino acid sequence of the Tub4 protein (Tub4p) is 29-38% identical to members of the gamma-tubulin family. Indirect immunofluorescence experiments using a strain containing an epitope-tagged Tub4p indicate that Tub4p resides at the spindle pole body throughout the yeast cell cycle. Deletion of the TUB4 gene indicates that Tub4p is essential for yeast cell growth. Tub4p-depleted cells arrest during nuclear division; most arrested cells contain a large bud, replicated DNA, and a single nucleus. Immunofluorescence and nuclear staining experiments indicate that cells depleted of Tub4p contain defects in the organization of both cytoplasmic and nuclear microtubule arrays; such cells exhibit nuclear migration failure, defects in spindle formation, and/or aberrantly long cytoplasmic microtubule arrays. These data indicate that the S. cerevisiae gamma-tubulin protein is an important SPB component that organizes both cytoplasmic and nuclear microtubule arrays.