Balance between Transcription and RNA Degradation Is Vital forSaccharomyces cerevisiaeMitochondria: Reduced Transcription Rescues the Phenotype of Deficient RNA Degradation

Abstract

The Saccharomyces cerevisiae SUV3 gene encodes the helicase component of the mitochondrial degradosome (mtEXO), the principal 3 -to-5 exoribonuclease of yeast mitochondria responsible for RNA turnover and surveillance. Inactivation of SUV3 (suv3⌬) causes multiple defects related to overaccumulation of aberrant transcripts and precursors, leading to a disruption of mitochondrial gene expression and loss of respiratory function. We isolated spontaneous suppressors that partially restore mitochondrial function in suv3⌬ strains devoid of mitochondrial introns and found that they correspond to partial loss-of-function mutations in genes encoding the two subunits of the mitochondrial RNA polymerase (Rpo41p and Mtf1p) that severely reduce the transcription rate in mitochondria. These results show that reducing the transcription rate rescues defects in RNA turnover and demonstrates directly the vital importance of maintaining the balance between RNA synthesis and degradation. INTRODUCTIONThe degradation of RNA is an essential element in the expression of genetic information. It is required to control RNA abundance and thus gene expression and to eliminate aberrant or defective molecules that inevitably form during RNA synthesis and maturation (RNA surveillance) (Vasudevan and Peltz, 2003). The posttranscriptional mechanisms affecting mitochondrial gene expression, including RNA turnover, are of particular importance because transcriptional control is relatively simple and rudimentary. The single RNA polymerase (RNAP) of Saccharomyces cerevisiae mitochondria is composed of only two nuclear-encoded protein subunits-the core enzyme encoded by the RPO41 gene and a transcription initiation factor encoded by the MTF1 gene (Masters et al., 1987;Jang and Jaehning, 1991). The RNAP holoenzyme recognizes a simple nonanucleotide promoter sequence (Osinga et al., 1982;Mangus et al., 1994) and initiates synthesis of at least 13 primary multicistronic transcripts that undergo extensive processing to form mature RNAs (Christianson and Rabinowitz, 1983;Tzagoloff and Myers, 1986;Foury et al., 1998;Gagliardi et al., 2004;Schafer, 2005). Such organization leaves little room for regulation at the transcription initiation level and makes posttranscriptional processes, including RNA degradation, key control points for mitochondrial gene expression.The enzymes controlling RNA turnover in cells and organelles show great evolutionary divergence and are only partially conserved between mitochondria of different organisms (Gagliardi et al., 2004). The enzymatic activity responsible for turnover is, however, on the basic level, similar in all the systems discovered so far-it is that of a 3Ј-to-5Ј processive exoribonuclease, either hydrolytic or phosphorolytic. In most cases, the exoribonuclease activity is contained in a larger multiprotein complex that, in addition to exoribonucleases, also contains RNA helicases, and in certain cases, endonucleases. A model example of such a complex is the eubacterial degradosome (Carpousis, 2002).The first mitoc...

DMR1 (CCM1/YGR150C) of Saccharomyces cerevisiae Encodes an RNA-Binding Protein from the Pentatricopeptide Repeat Family Required for the Maintenance of the Mitochondrial 15S Ribosomal RNA

“…At this moment it is not entirely clear whether this degradation is brought about by endonucleases, 59-39 exoribonucleases or a combination of endo-and exoribonucleolytic activities. The main ribonucleolytic activity of yeast mitochondria is assured by the mtEXO (mitochondrial degradosome) complex, which is a 39-59 exoribonuclease (Dziembowski et al 2003;Rogowska et al 2006;Malecki et al 2007), but the products of the degradation of 15S rRNA in the Figure 7.-15S rRNA transcripts in single and double pet127 and dmr1 deletants. Hybridization with an oligonucleotide probe recognizing the 39 fragment of the 15S rRNA shows that in the context of a r 1 mtDNA (intronless) the deletion of PET127 (pet127D strain DPET2) the 15S rRNA transcript is only slightly longer than in the isogenic and isomitochondrial control (CW252), but no other defects are apparent.…”

Yeast DEAD Box Protein Mss116p Is a Transcription Elongation Factor That Modulates the Activity of Mitochondrial RNA Polymerase

“…The results described above strongly suggested a role for Mss116p in mt transcription. To examine this further, we determined whether overexpression of genes that encode mt RNAP (RPO41) or the mt transcription initiation factor Mtf1p (MTF1) would suppress a deletion of MSS116, as had been observed for a deletion of SUV3 (as noted above, this observation was previously taken to suggest a role for SUV3 in balancing RNA synthesis and degradation/processing) (8,16). In earlier studies it had been shown that the deletion of MSS116 in an intron-less, mt splicing-dispensable strain (⌬mss116 I 0 ) led to a cold-sensitive growth defect on respirationdependent carbon sources at 22°C, suggesting that Mss116 has other roles in addition to its splicing-related functions.…”

“…Therefore, expression of nuclear and mt genes must be coordinately regulated (5, 6). As a consequence, mitochondria have developed a highly active RNA surveillance/degradation system to ensure the integrity of mt transcripts and to regulate the availability of mt mRNA by balancing the rates of RNA synthesis and degradation (7,8,9).Little is known about how transcription is controlled in mitochondria and what proteins participate in adjusting the rates of mt RNA synthesis and degradation. In the yeast Saccharomyces cerevisiae, mt transcription is carried out by an RNA polymerase (RNAP) consisting of a single-subunit catalytic core (Rpo41p) that is related to T7 RNA polymerase (T7 RNAP) and an additional transcription factor (Mtf1p) for initiation (10,11,12).…”

“…Therefore, expression of nuclear and mt genes must be coordinately regulated (5, 6). As a consequence, mitochondria have developed a highly active RNA surveillance/degradation system to ensure the integrity of mt transcripts and to regulate the availability of mt mRNA by balancing the rates of RNA synthesis and degradation (7,8,9).…”

“…RNA degradation in turn depends upon a two-subunit mt degradosome that includes Suv3p, a processive helicase that unwinds double-stranded RNA (dsRNA), and Dss1p, a 3= to 5= exonuclease (13,14). Importantly, deletion of SUV3 is suppressed by mutations in RPO41 and MTF1, suggesting that alterations in these components of the transcription system help to restore the balance between RNA synthesis and degradation (8). The SUV3 deletion is also suppressed by overexpression of Mss116p, an mt DEAD box protein that is involved in RNA splicing, processing, and possibly translation (15,16).…”