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Ccm1p is a 15S rRNA primary transcript processing factor as elucidated by a novel in vivo system in Saccharomyces cerevisiae

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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.

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taken from a master plate of YEPG-geneticin, grown in SDGal for 48 h (“On”; t = 0 h), and cultured on SSD (“Off” [filled downward pointing arrows]) and SDGal (“On” [unfilled upward pointing arrows]). b From the 72-h SDD culture (“Off”), cells were either grown in SDD (“Off” [filled downward pointing arrows] or SDGal (“Back on” [half filled upward pointing arrows]), as shown by the small horizontal arrow. White-dashed rectangles indicate the acquired transient mutant and wild-type phenotype during the entire experimental course

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Acknowledgements

This work was supported by the Mississippi INBRE funded by grants from the National Center for Research Resources [5P20RR016476] and the National Institute of General Medical Sciences (NIGMS) [8P20GM103476] from the National Institutes of Health (NIH), and by grants number [5SC3GM087169] and [W911NF-13-1-0174] from NIGMS, NIH, and DoD, respectively. We thank Mrs. Kimberley S. Buie for her contributions. The support from Dr. Sandra Barnes, Department of Chemistry and Physics, and the office of the Dean of the School of Art and Sciences are honestly appreciated. Finally, we acknowledge the valuable input from the reviewers.

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  1. Department of Biological Sciences, Alcorn State University, 1000 ASU Drive # 870, Lorman, MS, 39096, USA

    J. Ignacio Moreno, Ineshia S. Coleman, Classie L. Johnson, Dominique S. Green & Marta A. Piva

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  1. J. Ignacio Moreno
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  2. Ineshia S. Coleman
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Moreno, J.I., Coleman, I.S., Johnson, C.L. et al. Ccm1p is a 15S rRNA primary transcript processing factor as elucidated by a novel in vivo system in Saccharomyces cerevisiae. Curr Genet 66, 775–789 (2020). https://doi.org/10.1007/s00294-020-01064-0

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