RESUMO
Mammalian X inactivation, imprinting, and allelic exclusion are classic examples of monoallelic gene expression. Two emerging themes are thought to be critical for monoallelic expression: (1) noncoding, often antisense, transcription linked to differential chromatin marks on otherwise homologous alleles and (2) physical segregation of alleles to separate domains within the nucleus. Here, we highlight recent progress in identifying these phenomena as possible key regulatory mechanisms of monoallelic expression.
Assuntos
Núcleo Celular/genética , Regulação da Expressão Gênica/genética , Inativação Gênica/fisiologia , Perda de Heterozigosidade/genética , RNA não Traduzido/genética , Inativação do Cromossomo X/genética , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , DNA Antissenso/genética , Impressão Genômica/genética , Humanos , Transcrição Gênica/genéticaRESUMO
H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) are essential for the maturation and pseudouridylation of the precursor of rRNAs and other stable RNAs. Although the RNA and protein components of these RNPs have been identified, the mechanisms by which they are assembled in vivo are poorly understood. Here we show that the RNA binding protein Naf1p, which is required for H/ACA snoRNPs stability, associates with RNA polymerase II-associated proteins Spt16p, Tfg1p, and Sub1p and with H/ACA snoRNP proteins. Chromatin immunoprecipitation experiments show that Naf1p and the pseudouridylsynthetase Cbf5p cross-link specifically with the chromatin of H/ACA small nucleolar RNA (snoRNA) genes. Naf1p and Cbf5p cross-link predominantly with the 3' end of these genes, in a pattern similar to that observed for transcription elongation factor Spt16p. Cross-linking of Naf1p to H/ACA snoRNA genes requires active transcription and intact H/ACA snoRNA sequences but does not require the RNA polymerase II CTD kinase Ctk1p. These results suggest that Naf1p and Cbf5p are recruited in a cotranscriptional manner during H/ACA snoRNP assembly, possibly by binding to the nascent H/ACA snoRNA transcript during elongation or termination of transcription of H/ACA snoRNA genes.
Assuntos
Proteínas Fúngicas/metabolismo , Hidroliases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/fisiologia , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Hidroliases/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Fatores de Elongação da TranscriçãoRESUMO
Small nucleolar ribonucleoprotein particles (snoRNPs) are essential cofactors in ribosomal RNA metabolism. Although snoRNP composition has been thoroughly characterized, the biogenesis process of these particles is poorly understood. We have identified two proteins from the yeast Saccharomyces cerevisiae, Yil104c/Shq1p and Ynl124w/Naf1p, which are essential and required for the stability of box H/ACA snoRNPs. Depletion of either Shq1p or Naf1p leads to a dramatic and specific decrease in box H/ACA snoRNA levels in vivo. A severe concomitant defect in ribosomal RNA processing is observed, consistent with the depletion of this family of snoRNAs. Shq1p and Naf1p show nuclear localization and interact with Nhp2p and Cbf5p, two core proteins of mature box H/ACA snoRNPs. Shq1p and Naf1p form a complex, but they are not strongly associated with box H/ACA snoRNPs. We propose that Shq1p and Naf1p are involved in the early biogenesis steps of box H/ACA snoRNP assembly.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Endorribonucleases/metabolismo , Substâncias Macromoleculares , Processamento Pós-Transcricional do RNA , RNA Fúngico , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribonuclease III , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Previous molecular genetic studies on HeLa cell (a cervical cancer cell line) derived non-tumorigenic and tumorigenic hybrids have localized a tumor suppressor gene to the long arm of chromosome 11. Analysis of cervical cancer cell lines using chromosome 11 specific probes showed deletion and translocation of 11q13 sequences in five out of eight cell lines. Fluorescence in situ hybridization (FISH), using 11q13 specific probes, has shown interstitial deletion of 11q13 sequences in the HeLa cells. In order to determine whether 11q13 deletions occur in primary cervical tumors, we analysed 36 tumors using 20 different microsatellite and RFLP markers. Semi automated fluorescein based allelotyping was performed to identify loss of heterozygosity (LOH) in tumors. The results showed allelic loss in 17 (47%) tumors. Three different regions of loss, one near MEN1, the second near D11S913, and the third near INT2 locus were observed. The smallest region of deletion overlap at the D11S913 locus was localized to a 300 Kb distance between D11S4908 and D11S5023. Fluorescence in situ hybridization (FISH), using 11q13 specific cosmid and BAC (bacterial artificial chromosome) probes, confirmed allelic deletion in the tumors. PCR analysis further identified homozygous deletion of 11q13 sequences in a primary tumor, in HeLa cells and in two HeLa cell derived tumorigenic hybrid cell lines. The homozygous deletion in the cell lines was mapped to a 5.7 kb sequence of 11q13. We hypothesize therefore that a putative cervical cancer tumor suppressor gene exists within the 300 kb of chromosome 11q13.
