RESUMO
The RNA exosome complex processes and degrades a wide range of transcripts, including ribosomal RNAs (rRNAs). We used cryo-electron microscopy to visualize the yeast nuclear exosome holocomplex captured on a precursor large ribosomal subunit (pre-60S) during 7S-to-5.8S rRNA processing. The cofactors of the nuclear exosome are sandwiched between the ribonuclease core complex (Exo-10) and the remodeled "foot" structure of the pre-60S particle, which harbors the 5.8S rRNA precursor. The exosome-associated helicase Mtr4 recognizes the preribosomal substrate by docking to specific sites on the 25S rRNA, captures the 3' extension of the 5.8S rRNA, and channels it toward Exo-10. The structure elucidates how the exosome forms a structural and functional unit together with its massive pre-60S substrate to process rRNA during ribosome maturation.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/química , Exossomos/química , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/ultraestrutura , Complexo Multienzimático de Ribonucleases do Exossomo/ultraestrutura , Exossomos/ultraestrutura , Conformação Proteica , Precursores de RNA/química , Precursores de RNA/ultraestrutura , RNA Ribossômico/química , RNA Ribossômico/ultraestrutura , RNA Ribossômico 5,8S/química , RNA Ribossômico 5,8S/ultraestrutura , Ribossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Yeast large ribosomal subunit (LSU) precursors are subject to substantial changes in protein composition during their maturation due to coordinated transient interactions with a large number of ribosome biogenesis factors and due to the assembly of ribosomal proteins. These compositional changes go along with stepwise processing of LSU rRNA precursors and with specific rRNA folding events, as revealed by recent cryo-electron microscopy analyses of late nuclear and cytoplasmic LSU precursors. Here we aimed to analyze changes in the spatial rRNA surrounding of selected ribosomal proteins during yeast LSU maturation. For this we combined a recently developed tethered tertiary structure probing approach with both targeted and high throughput readout strategies. Several structural features of late LSU precursors were faithfully detected by this procedure. In addition, the obtained data let us suggest that early rRNA precursor processing events are accompanied by a global transition from a flexible to a spatially restricted rRNA conformation. For intermediate LSU precursors a number of structural hallmarks could be addressed which include the fold of the internal transcribed spacer between 5.8S rRNA and 25S rRNA, the orientation of the central protuberance and the spatial organization of the interface between LSU rRNA domains I and III.
Assuntos
RNA Ribossômico 5,8S/ultraestrutura , RNA Ribossômico/ultraestrutura , Subunidades Ribossômicas Maiores/ultraestrutura , Ribossomos/genética , Microscopia Crioeletrônica , Conformação de Ácido Nucleico , Biogênese de Organelas , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico 5,8S/química , RNA Ribossômico 5,8S/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores/química , Subunidades Ribossômicas Maiores/genética , Ribossomos/química , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genéticaRESUMO
Double target in situ hybridization to root tip metaphase and interphase cells of Silene cintrana and Silene rothmaleri was used to allocate the position of 18S-5.8S-25S and 5S rRNA genes. In both species, the 18S-5.8S-25S rDNA probe labelled four sites located on the short arms of two submetacentric chromosomes. Only one locus for 5S rDNA was mapped adjacent to 18S-5.8S-25S genes in a subterminal position on the centromere side: in S. rothmaleri the 5S rDNA locus was adjacent to the small 18S-5.8S-25S locus while in S. cintrana it was near the large one. The NOR activity analysed by Ag-staining in metaphase cells revealed proportionality between in situ labelling dimensions and Ag-NORs. In both species all rDNA loci were potentially active, although in S. rothmaleri a tendency for the expression of only one locus was observed. Interphase organisation analysis of rDNA showed some differences between both species that were correlated with NOR activity.
Assuntos
DNA Ribossômico/ultraestrutura , Interfase , Mapeamento Físico do Cromossomo , Raízes de Plantas/genética , Plantas/genética , Centrômero/ultraestrutura , Hibridização In Situ , Metáfase , Região Organizadora do Nucléolo/ultraestrutura , RNA Ribossômico 18S/ultraestrutura , RNA Ribossômico 5,8S/ultraestrutura , RNA Ribossômico 5S/ultraestrutura , Coloração pela PrataRESUMO
Diethyl pyrocarbonate reactivity and thermal denaturation were used to probe potential ribosomal interactions between tRNA and the small 5.8S and 5S rRNAs. Puromycin, an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, was observed to increase the accessibility of the 5.8S rRNA, including the highly conserved GAACp sequences. EDTA which releases both tRNA and the 5S rRNA-protein complex resulted in an even greater accessibility in the 5.8S rRNA. The thermal dissociation of whole ribosomes resulted in the release of all three RNAs, with a striking similarity in the denaturation profiles. These results strongly suggest an interdependence in the ribosome-associated structures of the small rRNAs and provide in situ evidence for the various 5S rRNA, 5.8S rRNA, and tRNA containing ribonucleoprotein complexes previously reconstituted through affinity chromatography.
