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

Zhu, Hong; Conrad-Webb, Heather; Liao, Xin; Perlman, Philip S.; Butow, Ronald A.

doi:10.1128/mcb.9.4.1507

Cited by 41 publications

(24 citation statements)

References 25 publications

(19 reference statements)

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“…Turnover of nascent RNAs was found to occur exonucleolyticaily; endonucleolytic cleavage products were not detected during turnover of the w intron RNA, which was studied in detail. (14,43,50). The levels of other classes of RNAs are not affected in this mutant.…”

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confidence: 76%

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A nucleoside triphosphate-regulated, 3' exonucleolytic mechanism is involved in turnover of yeast mitochondrial RNAs

Min

Zassenhaus

1993

J Bacteriol

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We have employed cell-free transcription reactions with mitochondria isolated from Saccharomyces cerevisiae to study the mechanism of RNA turnover. The specificity of RNA turnover was preserved in these preparations, as were other RNA-processing reactions, including splicing, 3' end formation of mRNAs, and maturation of rRNAs. Turnover of nascent RNAs was found to occur exonucleolyticaily; endonucleolytic cleavage products were not detected during turnover of the w intron RNA, which was studied in detail. (14,43,50). The levels of other classes of RNAs are not affected in this mutant. Other nuclear mutants specifically down regulate the stability of individual mRNAs (22,46). In the case of mutations in CBP1, which destabilize the cytb mRNA (17, 19), the protein encoded by the wild-type gene apparently interacts with RNA sequences in the 5' untranslated leader of the cytb mRNA (18,35).To understand how the specificity of RNA turnover is regulated in mitochondria, it is essential to know the mechanism by which RNAs are degraded. Little is known about this process in yeast cells, for either mitochondrial or cytoplasmic RNAs (8,24). From studies with other eukaryotes and bacteria, it is clear that both endo-and 3'-exoribonuclease activities participate in mRNA decay (for a review, see references 2 and 40). In several instances, for either eukaryotic or bacterial mRNAs the initial or rate-limiting step in mRNA turnover appears to be endonucleolytic scission of the transcript at discrete sites or regions within the RNA which are UA rich (1,4,12,26). Indeed, the relative paucity of UA dinucleotides in mRNAs has been attributed to the necessity to protect mRNAs from UpAselective RNases (3). Once cleaved, mRNA fragments are then rapidly degraded, apparently by the action of 3' exoribonucleases (20,44). Although in bacteria some mRNAs decay with an overall 5'-->3' polarity (25), this directionality is apparently not due to 5' exoribonucleolytic activity, since no such enzyme has been detected in bacteria (2). Instead, progressive 5'-3' endonucleolytic cuts along the transcript with subsequent 3' exonucleolytic degradation of the released 5' terminal fragments are thought to give rise to the observed 5'-3' polarity (9, 10).

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confidence: 76%

“…For example, a nuclear mutant has been characterized in which a variety of spliced group I introns accumulate to levels much higher than those in wild types (14,43,50). The levels of other classes of RNAs are not affected in this mutant.…”

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confidence: 99%

A nucleoside triphosphate-regulated, 3' exonucleolytic mechanism is involved in turnover of yeast mitochondrial RNAs

Min

Zassenhaus

1993

J Bacteriol

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“…Spheroplasts were washed twice in ice-cold mannitol buffer (0.6 M mannitol, 20 mM Tris-HCl [pH 7.4], 1 mM EDTA) and resuspended to 500 mg (wet weight)/ml in the same buffer containing protease inhibitors (24). After being chilled on ice, the suspension of spheroplasts was equilibrated with N2 gas (300 lb/in2) in a Parr bomb for 15 min and the spheroplasts were lysed by a quick release of the pressure (69). Mitochondria used in the preparation of mitoplasts were resuspended in freezing medium (29), frozen at -80°C, and thawed just before use.…”

Section: Materiasi and Methodsmentioning

confidence: 99%

HSP78 encodes a yeast mitochondrial heat shock protein in the Clp family of ATP-dependent proteases.

Leonhardt¹,

Fearson²,

Danese³

et al. 1993

Mol. Cell. Biol.

104

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The Saccharomyces cerevisiae nuclear gene for a 78-kDa mitochondrial heat shock protein (hsp78) was identified in a Agtll expression library through immunological screening with an hsp78-specific monoclonal antibody. Cells exposed to physiological stress, such as an abrupt elevation in temperature, synthesize a subset of highly conserved cellular proteins collectively known as the heat shock or stress proteins. Many members of the heat shock protein (HSP) families are expressed in the absence of stress and function as molecular chaperones in the folding of newly synthesized polypeptide chains and in the assembly of oligomeric structures (for recent reviews, see references 2 and 13). Heat shock proteins also appear to be involved in the degradation of unfolded or damaged polypeptides, either by catalyzing proteolysis or by presenting polypeptides to the active proteases. Well-known examples of heat-shockinducible proteins involved in protein degradation are ubiquitin in eukaryotes (55) and the ATP-dependent La (Lon) and ClpP proteases in prokaryotes (4,30). In addition, members of the newly discovered family of Clp proteins appear to combine an involvement in proteolysis with many of the characteristics of molecular chaperones, i.e., several are heat shock proteins, they are highly conserved in both prokaryotes and eukaryotes, and in eukaryotes, multiple forms or subfamilies appear to be partitioned in different cellular compartments.The wide distribution of the Clp family of proteins became apparent when the sequence of the ClpA subunit of the two-component ClpA-ClpP ATP-dependent protease (18), also known as the Ti protease (21), was found to be related to a number of previously described proteins with known sequences but unknown functions. These proteins, now * Corresponding author. t Present address:

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“…In this regard, a strain containing a mis-sense mutation in SUV3 (suv3-1) showed defects in 3Ј processing of mitochondrial mRNAs and increased stability of excised group I introns (9,18,19). In addition, inefficient splicing of aI5␤ and bI3, which are group I introns of COX1 and COB, respectively, was also observed.…”

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confidence: 99%

Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

Turk

Caprara

2010

Journal of Biological Chemistry

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Yeast Suv3p is a member of the DEXH/D box family of RNA helicases and is a critical component of the mitochondrial degradosome, which also includes a 3 3 5 exonuclease, Dss1p. Defects in the degradosome result in accumulation of aberrant transcripts, unprocessed transcripts, and excised group I introns. In addition, defects in SUV3 result in decreased splicing of the aI5␤ and bI3 group I introns. Whereas a role for Suv3p in RNA degradation is well established, the function of Suv3p in splicing of group I introns has remained elusive. It has been particularly challenging to determine if Suv3p effects group I intron splicing through RNA degradation as part of the degradosome, or has a direct role in splicing as a chaperone, because nearly all perturbations of SUV3 or DSS1 result in loss of the mitochondrial genome. Here we utilized the suv3-1 allele, which is defective in RNA metabolism and yet maintains a stable mitochondrial genome, to investigate the role of Suv3p in splicing of the aI5␤ group I intron. We provide genetic evidence that Mrs1p is a limiting cofactor for aI5␤ splicing, and this evidence also suggests that Suv3p activity is required to recycle the excised aI5␤ ribonucleoprotein. We also show that Suv3p acts indirectly as a component of the degradosome to promote aI5␤ splicing. We present a model whereby defects in Suv3p result in accumulation of stable, excised group I intron ribonucleoproteins, which result in sequestration of Mrs1p, and a concomitant reduction in splicing of aI5␤.Mitochondrial gene expression is largely controlled by posttranscriptional mechanisms because of the relatively simple nature of mitochondrial transcription (1-5). One critical control point is RNA degradation, which is involved in the extensive processing required of mitochondrial transcripts (6, 7). In Saccharomyces cerevisiae, intergenic regions are removed from polycistronic transcripts and in the case of two mRNAs, encoding the cytochrome oxidase I subunit (COX1) 2 and apocytochrome b subunit (COB), group I and group II catalytic introns are also removed. Splicing of these introns requires trans-acting factors to facilitate the RNA-catalyzed intron excision and ligation of flanking exons (8).Rapid turnover of excised group I introns is essential for mitochondrial gene expression because these RNAs act as endonucleases that cleave their parent, processed RNAs at the site of exon ligation (e.g. exon reopening reactions), which decreases expression (9). Degradation of excised group I introns is carried out by a two-protein complex called the mitochondrial degradosome or mtEXO (10, 11). The mitochondrial degradosome includes Dss1p, a 3Ј 3 5Ј single-stranded exonuclease, and Suv3p, a DEXH/D protein, one of the largest family of proteins that are involved in RNA metabolism (11-13). Indeed, some members show ATP-dependent unwinding of RNA helices. Suv3p is a member of the SKI2 subfamily, individuals of which are thought to couple ATP binding and hydrolysis cycles with translocation along an RNA single strand and disruption...

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

Cited by 41 publications

References 25 publications

A nucleoside triphosphate-regulated, 3' exonucleolytic mechanism is involved in turnover of yeast mitochondrial RNAs

A nucleoside triphosphate-regulated, 3' exonucleolytic mechanism is involved in turnover of yeast mitochondrial RNAs

HSP78 encodes a yeast mitochondrial heat shock protein in the Clp family of ATP-dependent proteases.

Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

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