Role of large subunit ribosomal protein L4 in ribosome maturation and cell cycle progression in Saccharomyces cerevisiae

Abstract

Eukaryotic ribosome biogenesis is a highly co‐coordinated multi‐step process that requires equimolar synthesis of several rRNAs and over 50 ribosomal proteins. Until recently, ribosomal proteins were mainly considered only as structural components of the ribosomes. This view has now changed with increasing evidence indicating consistent alterations in r‐protein expression levels during tumorogenesis and in certain developmental defects.Ribosomal protein L4 is a large ribosomal subunit protein that is structurally conserved in all kingdoms of life. Furthermore, there are several reports of alteration in regulation of L4 mRNA during tissue regeneration, interaction of L4 with transcriptional factors, and varied regulation of L4 mRNA in cancer cell lines. This indicates that L4 has key extra ribosomal functions along with its role in ribosomal biogenesis and function. Here we show that in vivo depletion of Rpl4p in S. cerevisiae results in severe loss of 60S ribosomal subunits. Northern blot, primer extension, and uracil pulse‐labeling experiments suggest this might be due to a block in the processing of the 27SA3 precursor RNA into 5.8S and 25S rRNA as well as a delay in processing of 35S precursor indicating an important role in maturation of the large subunit of the ribosome. More surprisingly, depletion of Rpl4 and select few large subunit ribosomal proteins results in accumulation of multiple budded phenotype. FACS analysis further confirms accumulation of 3N peak representing the multi budded phenotype. Initial microscopic analysis shows that the Rpl4p depleted cells might be getting arrested in late anaphase / early telophase with separated nuclei but disorganized micro tubules. Further experiments are being done to characterize this phenotype and to elucidate the role of Rpl4 in this particular cell cycle defect