Carbon catabolite repression of yeast CBP1 mRNA 3' end formation.

Abstract

CBP1 is a yeast nuclear gene encoding a mitochondrial protein that stabilizes the 5' end of cytochrome b (cob) pre-mRNA. Cytochrome b is the only mitochondrially synthesized component of the respiratory chain Complex III. Since the nuclearly-encoded subunits of this complex are regulated at the transcriptional level by catabolite repression, we hypothesized that CBP1 might be similarly regulated. To test the idea that transcriptional regulation of CBP1 could coordinate an increase in cytochrome b mRNA stability with an increase in nuclearly-encoded Complex III subunit production, we characterized the change in abundance of CBP1 mRNA during derepression on a non-fermentable carbon source. Poly A⁺ RNA from derepressed yeast was examined by Northern analyses with cRNA probes from CBP1. Both 2.2 kb and 1.3 kb transcripts were detected. The 1.3 kb mRNA lacks approximately 900 base-pairs of the 3'-end of the 2.2 kb mRNA, which encodes the carboxyl-terminal 250 amino acid residues of the CBP1 coding sequence. Northern analyses of RNA isolated from deletion/insertion mutants of CBP1 and from strains which overexpress CBP1 mRNA demonstrated that both mRNAs are transcribed from the CBP1 gene. Furthermore, we have demonstrated that the levels of the two CBP1 mRNAs are reciprocally regulated by the carbon source in the growth medium. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript we now show that a 146 bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2 kb mRNA, as mapped by sequence analysis of CBPl cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2 kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2 kb mRNA. Though none of the experiments showed whether the 1.2 kb mRNA is formed solely by 3' processing, or if processing at this site is coupled to premature transcription termination, our results do indicate that regulation of the abundance of both CBP1 transcripts is contro11ed by elements in a short segment of the gene that directs 3' end formation of the 1.2 kb transcript, a unique case in yeast.