Feedback Inhibition of the Yeast Ribosomal Protein Gene CRY2 Is Mediated by the Nucleotide Sequence and Secondary Structure of CRY2 Pre-mRNA

Abstract

The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells. Deletion or inactivation of CRY1 leads to 5-to 10-fold-increased levels of CRY2 mRNA. Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1. cis-Acting elements involved in repression of CRY2 were identified by assaying the expression of CRY2-lacZ gene fusions and promoter fusions in CRY1 CRY2 and cry1-⌬ CRY2 strains. Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5 exon and the first 62 nucleotides of the intron. Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression. The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5 end of the RP59 gene of the yeast Kluyveromyces lactis. Wild-type cells contain very low levels of both CRY2 pre-mRNA and CRY2 mRNA. Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm. Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally. A model for coupling ribosome assembly and regulation of ribosomal protein gene expression is proposed.The expression of ribosomal genes is coordinately regulated so that equimolar amounts of rRNAs and ribosomal proteins accumulate for assembly into ribosomes. The rate of synthesis of ribosomal molecules is also tightly coordinated with the physiological state of cells (reviewed in references 72 and 73). Coordinate synthesis of yeast ribosomal proteins is controlled primarily at the level of transcription of their genes through one of two common upstream activating sequences, UAS RPG or UAS T (reviewed in references 72 and 73). Posttranscriptional controls provide mechanisms for fine-tuning the expression of individual ribosomal protein (rp) genes (1,7,14,41,50,53,64,69).Balanced accumulation of ribosomal proteins in Escherichia coli results from feedback regulation of their expression (reviewed in references 47 and 48). Certain E. coli ribosomal proteins, when synthesized in excess of the rRNAs to which they bind in the assembling ribosome, repress expression of their own operons. Four eukaryotic rp genes have also been found to be autogenously regulated. Yeast ribosomal protein L32 binds to a structure comprising the 5Ј exon and the first few nucleotides of the intron of RPL32 pre-mRNA and blocks its splicing (15, 67). L32 also regulates the translation of its own mRNA through a similar secondary structure formed in the 5Ј end of the mature mRNA (8). Expression of the yeast rp gene RPL2 is autogenously controlled at the level of mRNA accumulation (53). The Xenopus laevis gene that encod...

Expression of a Micro-protein

“…In heterozygous diploid strains of genotype RPL41A/rpl41A or RPL41B/ rpl41B, the amount of L41A or L41B mRNA is reduced ϳ50%, respectively, as expected. Interestingly, in a haploid ⌬RPL41A strain the amount of L41B transcript increases about 35%, suggesting some measure of dosage compensation as has been shown for CRY2, encoding ribosomal protein S14 (32). No obvious change in the level of L41 mRNA was observed in a haploid ⌬RPL41B strain, perhaps reflecting the relatively larger amount of RPL41A transcript that is normally present.…”

Limited Portability of G-Patch Domains in Regulators of the Prp43 RNA Helicase Required for Pre-mRNA Splicing and Ribosomal RNA Maturation in Saccharomyces cerevisiae

“…The coordination of pre-mRNA processing with ribosome biogenesis is especially relevant in the intron-poor environment of the yeast genome, where the highly expressed ribosomal protein transcripts represent a disproportionate amount of the spliced mRNA (Ares et al 1999;Staley and Woolford 2009). Our understanding of how this coordination is accomplished is limited, however, to general principles supported by a few specific examples where individual ribosomal proteins act as feedback regulators to inhibit the processing or stability of cognate ribosomal protein transcripts (Li et al 1995(Li et al , 1996Vilardell et al 2000;Pleiss et al 2007;Gudipati et al 2012).…”

“…The coordination of pre-mRNA processing with ribosome biogenesis is especially relevant in the intron-poor environment of the yeast genome, where the highly expressed ribosomal protein transcripts represent a disproportionate amount of the spliced mRNA (Ares et al 1999;Staley and Woolford 2009). Our understanding of how this coordination is accomplished is limited, however, to general principles supported by a few specific examples where individual ribosomal proteins act as feedback regulators to inhibit the processing or stability of cognate ribosomal protein transcripts (Li et al 1995(Li et al , 1996Vilardell et al 2000;Pleiss et al 2007;Gudipati et al 2012).The chemistry of pre-mRNA splicing and the initial stages of rRNA processing occur in spatially separable nuclear Leeds et al 2006;Bohnsack et al 2009;Rodriguez-Galan et al 2013). The yeast Prp43 protein and its mammalian homolog DHX15 also act to dislodge the intron from the postcatalytic spliceosome and to recycle essential snRNP factors for use in subsequent rounds of splicing (Arenas and Abelson 1997;Martin et al 2002;Tsai et al 2005;Wen et al 2008;Fourmann et al 2013).…”

Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans

“…Only a handful of natural targets of mRNA surveillance have been identified to date. In yeast, unspliced cytoplasmic pre-mRNAs of CYH2, RP51B, MER2, and CRY2 contain stop codons within the retained introns and are efficiently eliminated by mRNA surveillance (He et al 1993;Li et al 1995). Certain mRNAs that undergo leaky scanning for translation initiation sites are also subject to mRNA surveillance in yeast (Welch and Jacobson 1999).…”