Analysis of pre-ribosomal processing and assembly factors in Saccharomyces cerevisiae

• Main Author: Thomson, Emma
• Format: Dissertation
• Language: English
• Published: University of Edinburgh 2006

To gain further insight into the pre-rRNA processing and maturation pathway, I identified three putative ribosome synthesis factors, NOP53, NOP9 and SDO1 through a bioinformatics approach. In cells depleted of Nop53p, synthesis of mature rRNA components of the 60S ribosomal subunit is severely inhibited. This aberration causes defective pre-60S subunits to accumulate in the nucleus. These particles are likely to contain the 7S and 25S’ pre-rRNAs, both of which strongly accumulate in a Nop53p depleted strain. Together this suggests that Nop53p is required for pre-60S particles to gain export competence. Nop53p has been identified as a protein that co-precipitated with Trf4p and Mtr4p, components of the TRAMP complex, which function together with the exosome in the surveillance and degradation of defective pre-ribosomes. It appears that transcripts which accumulate in Nop53p depleted cells are not subject to polyadenylation, which we hypothesise prevents efficient targeting of late pre-60S ribosomes for degradation by the TRAMP/exosome system. Nop9p is a nucleolar protein that co-purifies with the 20S pre-rRNA. On depletion of Nop9p the synthesis of mature 18S rRNA is severely inhibited due to a delay in early cleavage steps, whereby 20S fails to by synthesised.  Movement of 90S and/or pre-40S particles form the nucleolus to the nucleoplasm and cytoplasm is inhibited on depletion of Nop9p. Nop9p contains a pumilio-like RNA binding motif and in vitro binding assays demonstrate that Nop9p is capable of binding RNA. Sdo1p was identified as being required for the processing of the 35S pre-rRNA transcript. The human homologue of Sdo1p, SBDS, encodes a protein mutated in the marrow failure condition, Shwachman-Bodian Diamond Syndrome (SBDS). Depletion of Sdo1p results in a short delay in the synthesis of 5.8S rRNA.