Abstract
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, RPS22B and RPL1B, 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 of RPS22B is required for regulation, presumably because the protein inhibits splicing that is necessary for translation. The 5’ UTR of RPL1B contains a sequence and structure motif that is conserved in the binding sites of Rpl1 orthologs from bacteria to mammals, and mutations within the motif eliminate repression.
Highlights
Post-transcriptional autoregulation of gene expression is common in bacteria but many fewer examples are known in eukaryotes
We are interested in finding new examples of posttranscriptional autoregulation of protein expression so our initial focus is on ribosomal proteins, which are commonly translationally autoregulated in bacteria but[27,28,49,50] for which many fewer examples are known in yeast
RPS28B does not contain an intron but is autoregulated by a different mechanism where binding of the Edc[3] decapping enzyme to the 3′ UTR is regulated by the Rps[28] protein, leading to mRNA degradation[62,63]. These cases are all consistent with examples from bacteria where ribosomal protein synthesis is regulated post-transcriptionally, and it seems likely that a directed search for feedback regulation among yeast ribosomal protein genes could uncover more examples, leading us to utilize the yeast green fluorescent protein (GFP)-fusion collection
Summary
Post-transcriptional autoregulation of gene expression is common in bacteria but many fewer examples are known in eukaryotes. The collection of yeast strains with genes fused to green fluorescent protein (GFP)[48] provides an excellent resource to screen for examples of feedback regulation By introducing into those strains an inducible copy of a gene for the same protein, but lacking all potential cis-regulatory elements (cre-less), an observed reduction in the level of GFP after induction indicates some feedback mechanism controlling the expression of the wild-type gene. RPS28B does not contain an intron but is autoregulated by a different mechanism where binding of the Edc[3] decapping enzyme to the 3′ UTR is regulated by the Rps[28] protein, leading to mRNA degradation[62,63] These cases are all consistent with examples from bacteria where ribosomal protein synthesis is regulated post-transcriptionally, and it seems likely that a directed search for feedback regulation among yeast ribosomal protein genes could uncover more examples, leading us to utilize the yeast GFP-fusion collection
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