RESUMEN
Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo-electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.
Asunto(s)
Precursores del ARN/ultraestructura , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructura , Ribosomas/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN Espaciador Ribosómico/genética , ADN Espaciador Ribosómico/ultraestructura , Precursores del ARN/química , Precursores del ARN/genética , ARN Ribosómico/química , ARN Ribosómico/genética , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Ribosomas/química , Ribosomas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructuraRESUMEN
In order to investigate the ribosomal origin of the postsynaptic densities during chick cerebellar maturation, a new procedure for synaptosomal preparation was implemented. Samples from embryonic chick cerebellar cortex, from 14 to 20 days of development and young adult chicks, were initially mechanically dissociated after treatment with 1.25% trypsin for 10 min; with this procedure we were able to obtain a dendritic suspension from which an enriched synaptosomal fraction was prepared in a discontinuous Ficoll sucrose gradient. RNA and protein values determined from the synaptosomal fractions showed the following variations: from 46.3 ng RNA/mg of wet weight at day 14 to 616.7 ng RNA/mg of wet weight at day 18; in young adult chicks, the average was 242 ng RNA/mg of wet weight. RNA/protein ratio varied from 78.9 micrograms RNA/mg protein at day 16 to 329.8 micrograms RNA/mg protein at day 18; in the young adult chick, this ratio decreased to 68.4 micrograms RNA/mg protein. The highest value of RNA was obtained at day 18 of chick embryo development coinciding with the maximum period of synaptic formation and consequently of the PSDs. These results seem to reinforce the hypothesis of the ribosomal origin of the PSDs.