Synthesis and processing of ribosomal RNA in isolated yeast mitochondria

Boerner, Paula; Mason, Thomas L.; Fox, Thomas D.

doi:10.1093/nar/9.23.6379

Cited by 24 publications

(17 citation statements)

References 26 publications

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“…An appreciable portion of the decrease in mitochondrial RNA synthetic capacity in isolated organelles appears to be due to depression of rRNA synthesis, which accounts for only 10 to 20% of the total H-strand transcription (as estimated from densitometric measurements), instead of 85 to 90%, as is the case in vivo (4). The observed absolute rate of UMP incorporation into RNA of isolated HeLa cell mitochondria is comparable to the rates reported for isolated yeast mitochondria (6,14,22).…”

Section: Discussionsupporting

confidence: 53%

“…Furthermore, the in vivo-and in vitrosynthesized transcripts starting at this initiation site have been found to extend beyond the 3' end of 7S RNA for up to more than 600 NTPs (Shuey and Attardi, in preparation). These results have suggested the possibility that these transcripts may be a part of the putative fast-turning-over whole L-strand polycistronic transcripts destined to yield by processing the eight tRNA species and the mRNA for the unidentified reading frame (URF) 6 encoded in the L strand (26). According to this view, 7S RNA may also derive from processing of these polycistronic transcripts; alternatively, this RNA species may be independently transcribed, starting at a site very close to or even coinciding with the initiation site so far identified.…”

Section: Discussionmentioning

confidence: 99%

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Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

Gaines¹,

Attardi²

1984

Mol. Cell. Biol.

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A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37°C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNAsynthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmicmitochondrial interactions in organelle gene expression.Recent work from this laboratory has shown that the heavy (H) strand of HeLa cell mitochondrial DNA (mtDNA) is the site of two overlapping transcription events (4a, 12, 19. 20). A considerable amount of evidence suggests that one of these events is responsible for the synthesis of the bulk of rRNA, whereas the other results in the synthesis of a polycistronic molecule which corresponds to almost the entire H strand and is destined to be processed to yield the mRNAs and most of the tRNAs encoded in the H strand (Fig. 1). The initiation of the two H-strand transcription events and that of light (L)-strand transcription occur in close proximity to each other in the region of mtDNA upstream of the origin of H-strand synthesis (10). This clustering of the three putative promoters may underlie the integrated control of expression of the corresponding transcription units, whereas their proximity to the origin of Hstrand synthesis may reflect the existence of a link between transcription events and mtDNA replication. To investigate further, under conditions allowing easy experimental manipulations, the molecular mechanisms, controls, and interrelationships of the mtDNA transcription and RNA processing events which occur in HeLa cell mitochondria, we developed a system f...

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Section: Discussionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

Gaines¹,

Attardi²

1984

Mol. Cell. Biol.

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“…RNase T1 digestion of oligo 5-RNA heteroduplexes yielded not only the predicted fragment ( FIG. 5. Turnover of the w intron requires NTP.…”

Section: Methodsmentioning

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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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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“…extension, which is removed by a nuclease to generate the mature 5Ј-end (35,36). The 15 S rRNA precursor accumulates in pet127 mutants as do precursor transcripts of cytochrome b (COB) and VAR1, which are also processed in their 5Ј regions (37,38).…”

Section: Mtg3-dependent Processing Of Yeast Mitochondrial 15 S Rrnamentioning

confidence: 99%

The Putative GTPase Encoded by MTG3 Functions in a Novel Pathway for Regulating Assembly of the Small Subunit of Yeast Mitochondrial Ribosomes

Paul

Alushin

Barros

et al. 2012

Journal of Biological Chemistry

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Background: Assembly of the 54 S subunit of yeast mitochondrial ribosomes is assisted by two GTPases encoded by MTG1 and MTG2. Results: Processing of the 15 S rRNA precursor is blocked in mtg3 mutants. Conclusion: MTG3 codes for a putative GTPase that regulates processing and assembly of the 15 S rRNA precursor. Significance: The pathways for biogenesis of fungal mitochondrial and bacterial ribosomes employ conserved GTPases.

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Synthesis and processing of ribosomal RNA in isolated yeast mitochondria

Cited by 24 publications

References 26 publications

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

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

The Putative GTPase Encoded by MTG3 Functions in a Novel Pathway for Regulating Assembly of the Small Subunit of Yeast Mitochondrial Ribosomes

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