Pet127 Governs a 5′ → 3′-Exonuclease Important in Maturation of Apocytochrome b mRNA in Saccharomyces cerevisiae

Abstract

The details of mRNA maturation in Saccharomyces mitochondria are not well understood. All seven mRNAs are transcribed as part of multigenic units. The mRNAs are processed at a common 3-dodecamer sequence, but the 5-ends have seven different sequences. To investigate whether apocytochrome b (COB) mRNA is processed at the 5-end from a longer precursor by an endonuclease or an exonuclease, a 64-nucleotide sequence, which is required for the protection of COB mRNA by the Cbp1 protein and is found at the 5-end of the processed COB mRNA, was duplicated in tandem. The wild-type 64-nucleotide element functioned in either the upstream or downstream position when paired with a mutant element. In the tandem wild-type strain, the 5-end of the mRNA was at the 5-end of the upstream unit, demonstrating that the mRNA is processed by an exonuclease. Accumulation of precursor COB RNA in single and double element strains with a deletion of PET127 demonstrated that the encoded protein governs the 5-exonuclease responsible for processing the precursor to the mature form.In Saccharomyces cerevisiae, mitochondrial apocytochrome b (COB) 3 mRNA is transcribed from the tRNA glu -COB operon and is extensively processed before it reaches its mature form. Many aspects of this process have been well described; at least 15 nuclearly encoded proteins have been identified that control the metabolism of COB mRNA. As depicted in Fig. 1, the initial COB precursor is transcribed from position Ϫ1566, with the A of the AUG codon of COB defined as ϩ1, and contains both tRNA glu and COB mRNA (1, 2). Mitochondrial RNase P and a tRNA 3Ј-endonuclease cleave the initial precursor at positions Ϫ1170 and Ϫ1098, respectively, releasing tRNA glu from the precursor mRNA (3, 4). The mature mRNA is generated by further processing of the precursor RNA at position Ϫ955 or Ϫ954 (5). This trimming step and stabilization of the mature mRNA are not completely understood; however, it is known that the nuclearly encoded protein Cbp1 specifically controls the turnover (5-9) and translation (10) of COB mRNA. In cbp1 mutant strains, the level of tRNA glu is close to that of wild type, whereas precursor COB mRNA is reduced to 25% of wild-type levels and mature COB mRNA is undetectable.The best candidate for the mitochondrial nuclease that processes the COB precursor at Ϫ954/Ϫ955 is a mitochondrial membrane-bound protein, Pet127 (11). It is a member of a large protein family that is well conserved in fungi and protists but is not present in plants or mammals (12). In pet127 strains, 5Ј-end processing of COB, VAR1, and ATP8/6 mRNAs, 15 S rRNA (11), and RPM1, the mitochondrial RNase P RNA 4 (13) is blocked and unprocessed precursor RNAs accumulate to levels equivalent to the sum of processed and unprocessed RNAs in wild-type strains. These defects in processing only inhibit respiratory growth when the temperature is raised to 37°C (11). Pet127 could itself be the RNase that trims these 5Ј-ends, but the protein has no RNase signature sequences or domains. Alternatively, Pet127 c...

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

“…The putative 59-39 exoribonuclease Pet127p may be involved in the degradation of 15S rRNA in the absence of Dmr1p: The only known mitochondrial 59-39 exoribonucleolytic activity depends on the protein encoded by the PET127 gene (Wiesenberger and Fox 1997;Fekete et al 2007). As the deletion of DMR1 results in fragmentation of 15S rRNA, with the 39 end left intact, Pet127p is an obvious candidate for the RNase activity responsible for this degradation.…”

“…absence of Dmr1p do contain an intact 39 region. The only known mitochondrial 59-39 exo activity is governed by the Pet127 protein (Wiesenberger and Fox 1997;Fekete et al 2007), and the absence of distinct 15S rRNA degradation products in the pet127D dmr1D strain may suggest that Pet127p is indeed responsible for the degradation. It should, however, be noted that the deletion of PET127 in the context of the r À mitochondrial DNA results in a significant perturbation in the processing of the 15S rRNA precursors, which can obfuscate the phenotype of the deletion of DMR1.…”

tRNA 3′ processing in yeast involves tRNase Z, Rex1, and Rrp6

“…Nuc1 is a major mitochondrial nuclease with RNase and DNase activities (Vincent et al 1988), Pet127 is probably the main nonspecific 5 ′ -3 ′ exonuclease in mitochondria and is involved in 5 ′ processing of several mtRNAs, including RPM1 (Wiesenberger and Fox 1997;Ellis et al 2005;Fekete et al 2008;Schonauer et al 2008), whereas 3 ′ -5 ′ exonuclease Rex2, in addition to its functions in the nucleus, was shown to have mitochondrial localization (Hanekamp and Thorsness 1999;Kumar et al 2002;Huh et al 2003). In addition, physical or genetic interactions were demonstrated between Trz1 and Nuc1 or Rex1, respectively (Chen et al 2005;Benschop et al 2010).…”

Partitioning of the Nuclear and Mitochondrial tRNA 3′-End Processing Activities between Two different Proteins in Schizosaccharomyces pombe

“…In contrast, mtRNA processing in S. cerevisiae is a complex process (19). After tRNA processing, which liberates the 5Ј-and 3Ј-ends of pre-mRNAs and pre-rRNAs, additional processing steps are required for the generation of mature mRNAs and rRNAs in S. cerevisiae (20).…”