The High-resolution Timeline of Expression of Ribosomal Protein Genes in Yeast (original) (raw)
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Ribosome queuing is a fundamental phenomenon suggested to be related to topics such as genome evolution, synthetic biology, gene expression regulation, intracellular biophysics, and more. However, this phenomenon hasn't been quantified yet at a genomic level. Nevertheless, methodologies for studying translation (e.g. ribosome footprints) are usually calibrated to capture only single ribosome protected footprints (mRPFs) and thus limited in their ability to detect ribosome queuing. On the other hand, most of the models in the field assume and analyze a certain level of queuing. Here we present an experimental-computational approach for studying ribosome queuing based on sequencing of RNA footprints extracted from pairs of ribosomes (dRPFs) using a modified ribosome profiling protocol. We combine our approach with traditional ribosome profiling to generate a detailed profile of ribosome traffic. The data are analyzed using computational models of translation dynamics. The approach...
Molecular and Cellular Biology, 1986
The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also sugges...
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Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to c...
Autoregulation of yeast ribosomal proteins discovered by efficient search for feedback regulation
Communications Biology
Post-transcriptional autoregulation of gene expression is common in bacteria but many fewer examples are known in eukaryotes. We used the yeast collection of genes fused to GFP as a rapid screen for examples of feedback regulation in ribosomal proteins by overexpressing a non-regulatable version of a gene and observing the effects on the expression of the GFP-fused version. We tested 95 ribosomal protein genes and found a wide continuum of effects, with 30% showing at least a 3-fold reduction in expression. Two genes,RPS22BandRPL1B, showed over a 10-fold repression. In both cases the cis-regulatory segment resides in the 5’ UTR of the gene as shown by placing that segment of the mRNA upstream of GFP alone and demonstrating it is sufficient to cause repression of GFP when the protein is over-expressed. Further analyses showed that the intron in the 5’ UTR ofRPS22Bis required for regulation, presumably because the protein inhibits splicing that is necessary for translation. The 5’ UTR...
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Nucleic acids …, 1987
Transcription of the gene encoding yeast ribosomal protein L25 was previously shown to be activated through tandemly arranged upstream sequence elements that most rp-genes in yeast have in common. A single copy of such a conserved element is now demonstrated to restore transcription of an inactivated heterologous gene, which confirms its role as a genuine UAS: UAS . Though a single box is sufficient to activate transcription, most rp-genes harbor two neigouring elements. Northern analysis of mutants of the L25 upstream region lacking either the gene-distal (RPG1) or the gene-proximal (RPG2) box provided evidence that RPG2 is significantly more effective than RPG1 in vivo. Moreover the sum of the effects of the individual boxes as measured separately is significantly lower than their joint effect, supporting cooperative interaction between the two boxes in vivo. Making use of oligomer-insertion experiments several additional features of the UAS were elucidated. First of all we confirmed that the extent of transcription activation by the MgS depends upon the orientation of the element. Secondly we show that a certain minimal disA&nce (> 100 n) between UAS and the transcription initiation site is required for transcription activation. Finally, internal dele}l&n of the L25-upstream region as well as oligomer-insertion shed some light on the nucleotide requirements of the UAS pg.
Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae
Genetics, 2013
Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structur...
Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation
RNA, 2004
The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA 3 to 27SB S pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA 3 and 27SB L pre-ribosomal RNAs.
PLOS ONE, 2015
Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins) and ribosomal RNAs (rRNAs). Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs) with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU) rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs.