Ccm1p is a 15S rRNA primary transcript processing factor as elucidated by a novel in vivo system in Saccharomyces
J Ignacio Moreno1, Ineshia S Coleman2, Classie L Johnson2
1Department of Biological Sciences, Alcorn State University, 1000 ASU Drive # 870, Lorman, MS, 39096, USA. jmoreno@alcorn.edu.
Abstract:
In Saccharomyces cerevisiae, the mitoribosomal RNA of the minor subunit, 15S rRNA, is transcribed as a bicistronic transcript along with tRNAW. 5' and 3' sequences flanking the mature transcript must be removed by cleavage at the respective junctions before incorporating it into the mitoribosome. An in vivo dose-response triphasic system was created to elucidate the role of Ccm1p in the processing of 15S rRNA: Ccm1p supply ("On"), deprivation ("Off"), and resupply ("Back on"). After 72 h under "Off" status, the cells started to exhibit a complete mutant phenotype as assessed by their lack of growth in glycerol medium, while keeping their mitochondrial DNA integrity (ρ+). Full functionality of mitochondria was reacquired upon "Back on." 15S rRNA levels and phenotype followed the Ccm1p intramitochondrial concentrations throughout the "On-Off-Back on" course. Under "Off" status, cells gradually accumulated unprocessed 5' and 3' junctions, which reached significant levels at 72-96 h, probably due to a saturation of the mitochondrial degradosome (mtEXO). The Ccm1p/mtEXO mutant (Δccm1/Δdss1) showed a copious accumulation of 15S rRNA primary transcript forms, which were cleaved upon Ccm1p resupply. The gene that codes for the RNA component of RNase P was conserved in wild-type and mutant strains. Our results indicate that Ccm1p is crucial in processing the 15S rRNA primary transcript and does not stabilize the already mature 15S rRNA. Consequently, failure of this function in Δccm1 cells results, as it happens to any other unprocessed primary transcripts, in total degradation of 15S rRNA by mtEXO, whose mechanism of action is discussed.
Insights
Ccm1p is essential for processing the 15S rRNA primary transcript in Saccharomyces cerevisiae mitochondria. Its absence leads to degradation by mtEXO, impacting mitochondrial function.
Area of Science:
- Mitochondrial biology
- Molecular genetics
- RNA processing
Background:
- Mitoribosomal RNA (15S rRNA) requires processing of flanking sequences for function.
- Ccm1p's role in 15S rRNA maturation in Saccharomyces cerevisiae was previously unclear.
Purpose of the Study:
- To elucidate the function of Ccm1p in 15S rRNA processing.
- To investigate the consequences of Ccm1p deprivation and resupply on mitochondrial function and rRNA levels.
Main Methods:
- An in vivo triphasic system (On-Off-Back on) was used to control Ccm1p levels.
- Phenotypic analysis (growth on glycerol) and molecular analysis (15S rRNA levels, unprocessed junctions) were performed.
- A Ccm1p/mtEXO double mutant was analyzed.
Main Results:
- Ccm1p deprivation led to a loss of mitochondrial function and accumulation of unprocessed 15S rRNA precursors.
- Unprocessed junctions accumulated significantly after 72-96 hours of Ccm1p absence, suggesting mtEXO saturation.
- Ccm1p resupply restored mitochondrial function and processing of accumulated transcripts.
- The Ccm1p/mtEXO mutant showed massive accumulation of primary transcripts.
Conclusions:
- Ccm1p is crucial for the initial processing of the 15S rRNA primary transcript.
- Ccm1p does not stabilize mature 15S rRNA; its absence leads to degradation by mtEXO.
- The study clarifies Ccm1p's role in preventing degradation of unprocessed mitochondrial transcripts.
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