5 AND 3 END PROCESSING OF YEAST MITOCHONDRIAL PRE-MRNAS

Mitochondrial biogenesis requires the coordinate expression of the nuclear
and mitochondrial genomes. The long term goal is to understand the nature
of the controlling signals in Saccharomyces cerevisiae that synchronize the
production of mitochondrial constituents encoded in the separate
compartments. The characterization of nuclear respiratory deficient
strains of yeast, pet mutants, has identified several nuclear genes
necessary for proper processing of mitochondrial transcripts and
translation of the mRNAs. The aim of this proposal is to investigate the
role of two nuclearly encoded mitochondrial RNA processing enzymes in
regulating the synthesis of mitochondrially encoded proteins. A nuclear
gene product, initially identified by pet mutations, is specifically
responsible for conferring a stable 5' terminus on the mitochondrial mRNA
for cytochrome b, the only mitochondrially encoded subunit of coenzyme Q -
cytochrome c reductase. This CBP1, (cytochrome b processing), protein will
be further purified by column chromatography, and its action will be
studied in vitro. RNAs produced in an SP6 bacteriophage
promoter-transcription system will be used as substrates. The sequence and
structural requirements of the RNA substrate for recognition by the protein
will be investigated by characterizing mitochondrial suppressors that
alleviate cbp1 mutations, and by testing altered RNA substrates in the in
vitro assay. Another protein will be isolated that is responsible for
cleaving mitochondrial multigenic primary transcripts into unigene
segments, thereby conferring mature 3' termini on several mitochondrial
mRNAs. Purification of this endonuclease will be accomplished by one of
two approaches. The protein will be purified by conventional
chromatographic methods, employing an in vitro functional assay with
artificially SP6-generated RNA substrates. Using antibodies raised to this
protein, the nuclear gene encoding the enzyme will be selected from a cDNA
expression library. Alternatively, an indirect approach will be used,
involving characterization of pet mutants defective in this activity,
isolation of the gene by transformation with cloned wild-type yeast DNA,
production of an antibody to an overexpressed E. coli trp E-3'processing
gene fusion product, and immuno-detection of the protein throughout a
chromatographic procedure. To ascertain whether the genes encoding these
proteins are under a higher order regulatory system that coordinates
several nuclearly encoded mitochondrial components simultaneously, changes
in the levels of mRNAs produced for the specific 5'-end and general 3'-end
endonucleases will be monitored when wild-type yeast is switched from
glucose repression conditions to growth on a non-fermentable carbon source.