Expression of the split gene cob in yeast: Evidence for a precursor of a “maturase” protein translated from intron 4 and preceding exons

The 3' ends of most Saccharomyces cerevisiae mitochondrial mRNAs terminate at a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3', of unknown function. We have studied the consequences of mutations within a dodecamer found in an 1,143-base-pair optional intron of the mitochondrial large (21S) rRNA gene on RNA processing. The dodecamer is situated at the 3' end of an expressed open reading frame (ORF) within that intron, and the mutations are two adjacent transversions that extend the intron ORF by 51 nucleotides. The strain harboring these mutations, L5-10-1, is defective in biased intron transmission in crosses to strains that lack the intron, as are other mutants which contain nucleotide changes within the ORF (I. G. Macreadie, R. M. Scott, A. R. Zinn, and R. A. Butow, Cell 41:395-402, 1985). However, unlike these other mutants, wild-type strains, or petites which retain the intron allele, L5-10-1 is defective in processing at the intron dodecamer. In addition, L5-10-1 lacks a prominent 2.7-kilobase RNA containing both intron and exon sequences and at least two of four RNAs that correspond to various forms of the excised intron. We propose that these RNAs, missing in L5-10-1 but present in al other strains examined, arise in part by processing at the intron dodecamer. In addition, in all strains examined, we have detected a novel processing activity in which precursor 21S rRNA transcripts are cleaved in the upstream exon, about 1,500 nucleotides from the 5' end of the RNA. This activity, together with 3' intron dodecamer cleavage, probably accounts for the 2.7-kilobase RNA species, a candidate for the mRNA for the intron-encoded protein.Mature transcripts of the mitochondrial genome of Saccharomyces cerevisiae are generated by diverse pathways. Some genes contain a highly conserved nonanucleotide sequence at their 5' end that functions as an initiator for mitochondrial RNA polymerase (7,29). Other genes are cotranscribed into polycistronic precursors that are subsequently processed to yield the mature RNAs (6, 9, 23, 37). At the 3' end of almost all yeast mitochondrial protein structural genes is a highly conserved dodecamer sequence, 5'-AATAATATTCTT-3', which is located either between genes within a single transcription unit or at the 3' end of a transcription unit. In either case, the putative mRNAs for these genes terminate within the dodecamer sequence, most likely as a result of processing (30,33). The role of this 12-mer in mRNA activity or stability is unknown.A conserved dodecamer is also found within an optional 1,143-base-pair (bp) intron in the 21S rRNA gene but in an unusual location; it is at the 3' end of a freestanding open reading frame (ORF) (13) located entirely within the intron, and the termination codon for that ORF is part of the dodecamer sequence. The 21S rRNA intron is unique among optional mitochondrial introns, because in genetic crosses between strains that have it (wX) and those lacking it (w-), almost all of the w-alleles are converted to w+ (14,40 when crosses are carried out betw...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

RNA processing and expression of an intron-encoded protein in yeast mitochondria: role of a conserved dodecamer sequence.

Zhu¹,

Macreadie²,

Butow³

1987

“…Since the fit) gene is contained entirely within the intron itself (7), unlike the case with most other intron-encoded proteins of the yeast mitochondrial genome (8,19), the dramatic increase in the amount of excised intron (at least 40-fold) in the SUV3-1 background could, in principle, provide a source of message for the intron-encoded endonuclease. Moreover, it appears that, as with other intron-encoded proteins, only small amounts of the product are required for wild-type function.…”

Section: Strainsmentioning

confidence: 99%

Functional expression of a yeast mitochondrial intron-encoded protein requires RNA processing at a conserved dodecamer sequence at the 3' end of the gene.

Zhu¹,

Conrad-Webb²,

Liao³

et al. 1989

All mRNAs of yeast mitochondria are processed at their 3' ends within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3'. A dominant nuclear suppressor, SUV3-1, was previously isolated because it suppresses a dodecamer deletion at the 3' end of the varl gene. We have tested the effects of SUV3-1 on a mutant containing two adjacent transversions within a dodecamer at the 3' end of fit), a gene located within the 1,143-base-pair intron of the 21S rRNA gene, whose product is a site-specific endonuclease required in crosses for the quantitative transmission of that intron to 21S alleles that lack it. Thefit) dodecamer mutations blocked both intron transmission and dodecamer cleavage, neither of which was suppressed by SUV3-1 when present in heterozygous or homozygous configurations. Unexpectedly, we found that SUV3-1 completely blocked cleavage of the wild-typefit) dodecamer and, in SUV3-1 homozygous crosses, intron conversion. In addition, SUV3-1 resulted in at least a 40-fold increase in the amount of excised intron accumulated. Genetic analysis showed that these phenotypes resulted from the same mutation. We conclude that cleavage of a wild-type dodecamer sequence at the 3' end of the fit) gene is essential for fit) expression.The 3' ends of all mRNAs of yeast mitochondria are formed by endonucleolytic processing within a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3' of unknown function (12,17,20). Recently, two mutants involving dodecamers have been analyzed whose properties support the contention that 3'-end processing at dodecamer sequences is important for mitochondrial gene expression.The first mutant, L5-10-1, contains two adjacent transversions within the dodecamer offitl, a gene located within the 1,143-base-pair (bp) optional intron [omega] of the 21S rRNA gene (9). fit] encodes a site-specific endonuclease required in crosses for the quantitative transmission of [omega] into 21S rRNA alleles that lack it (2, 9, 22). We have shown that the L5-10-1 mutations completely block intron conversion as well as cleavage at the intron dodecamer, which indicates that the wild-type sequence is required there for 3' processing (9, 21). Pre-21S rRNA splicing and the abundance (stability) of excised intron RNA are unaffected by the mutations. Because the dodecamer mutations also extend the fit) reading frame, we could not conclude whether the intron transmission defect results from synthesis of a nonfunctional protein, the absence of thefitlmRNA, or both.The other mutant, PZ206, is a deletion of a dodecamer plus 195 bp of flanking sequences at the 3' end of the varl gene (H. M. Conrad-Webb and P. S. Perlman, manuscript in preparation). In the mutant, transcripts containing the varl reading frame are of low abundance and most terminate at either of two sequences, unrelated to the dodecamer, downstream of the deletion. Although the mutation does not alter the integrity of the varl reading frame, it blocks varl gene expression. ponent of the mitochondrial small ribosomal subunit (15, 16), PZ206 is severely deficient in...

“…Introns of the yeast mitochondrial cytochrome b gene were shown to contain long open reading frames which code for a maturase involved in the splicing of its introns as well as introns of other mitochondrial genes (33,60). The identification of dominant mutations in the cytochrome b introns that are far removed from the splice junctions indicates the existence of additional sequences needed for the removal of introns (2,17,40).…”

mentioning

confidence: 99%

Role of intron-contained sequences in formation of moloney murine leukemia virus env mRNA.

Hwang¹,

Park²,

Gilboa³

1984

Formation of the Moloney murine leukemia virus envelope mRNA involves the removal of a 5,185-base pairlong intron. Deletion analysis of two Moloney murine leukemia virus-derived expression vectors revealed the existence of two short regions within the viral intron which are required for the efficient formation of the spliced RNA species. One region was present upstream from the 3' splice junction, extended at least 85 nucleotides beyond the splice site, and was not more than 165 nucleotides long. As yeast polymerase II introns, the Moloney murine leukemia virus intron contains the sequence 5'-TACTAAC-3' 15 nucleotides upstream from the 3' splice site. A second region located in the middle of the intron, within a 560-nucleotide-long sequence, was also essential for formation of the spliced RNA species. The efficient splicing of the env mRNA in the absence of expression of viral genes raises the possibility that similar mechanisms are used to remove introns of (some) cellular genes.The coding regions of many eucaryotic genes are interrupted by stretches of noncoding DNA called intervening sequences or introns. The introns are transcribed as part of a precursor RNA and are subsequently removed in a process termed RNA splicing. A central problem in understanding the mechanism of splicing is identification of the RNA sequences responsible for the accurate removal of introns.Study of naturally occurring mutations and site-specific mutagenesis has shown that the nucleotides immediately adjacent to the splice junctions are required for splicing to occur. Deletions which removed the splice junctions of simiam virus 40 (SV40) small t antigen (27) or SV40 late mRNA (30, 55) abolished the synthesis of these RNA species. Similarly, globin genes from human thalassemic patients which contained mutations at the splice junctions did not generate the normally spliced RNA species (19,52,53). Site-specific mutagenesis has also shown that the nucleotides adjacent to the splice junctions are necessary for the formation of correctly spliced RNA species (20,37,61). The sequences adjacent to the splice junctions are strongly conserved, further demonstrating their functional importance (6,38,47 A large body of evidence indicates that the cis-acting regions required for efficient removal of introns exert their function through the formation of a secondary structure which brings the 5' and 3' splice junctions in close proximity. On the basis of nucleotide sequences of introns derived from fungal mitochondria and tetrahymena nuclear rRNA as well as Z. mays chloroplast mRNA, similar models of secondary structures that facilitate intron removal were proposed (10,16,36). Direct evidence for the role of a secondary structure in intron removal is provided by physical studies of the N. crassa mitochondrial rRNA intron (63), in vitro mutagenesis of the yeast tRNAleU intron (4), and analysis of cis mutations and their revertants in the yeast mitochondrial cytochrome b intron (59).Evidence for the role of sequences other than the consensus sequences...