Intersection of RNA Processing and the Type II Fatty Acid Synthesis Pathway in Yeast Mitochondria

Abstract

Distinct metabolic pathways can intersect in ways that allow hierarchical or reciprocal regulation. In a screen of respiration-deficient Saccharomyces cerevisiae gene deletion strains for defects in mitochondrial RNA processing, we found that lack of any enzyme in the mitochondrial fatty acid type II biosynthetic pathway (FAS II) led to inefficient 5 processing of mitochondrial precursor tRNAs by RNase P. In particular, the precursor containing both RNase P RNA (RPM1) and tRNA Pro accumulated dramatically. Subsequent Pet127-driven 5 processing of RPM1 was blocked. The FAS II pathway defects resulted in the loss of lipoic acid attachment to subunits of three key mitochondrial enzymes, which suggests that the octanoic acid produced by the pathway is the sole precursor for lipoic acid synthesis and attachment. The protein component of yeast mitochondrial RNase P, Rpm2, is not modified by lipoic acid in the wild-type strain, and it is imported in FAS II mutant strains. Thus, a product of the FAS II pathway is required for RNase P RNA maturation, which positively affects RNase P activity. In addition, a product is required for lipoic acid production, which is needed for the activity of pyruvate dehydrogenase, which feeds acetyl-coenzyme A into the FAS II pathway. These two positive feedback cycles may provide switch-like control of mitochondrial gene expression in response to the metabolic state of the cell.Intersections of biological pathways previously thought to be distinct have emerged recently from genome-wide functional screens in model organisms (52,62,63). Through a genomewide screen of Saccharomyces cerevisiae, we have discovered the intersection of mitochondrial type II fatty acid synthesis (FAS II) with mitochondrial RNA processing.There are two distinct fatty acid synthesis pathways in eukaryotes. Fatty acid synthesis type I (FAS I) is catalyzed by cytosolic multifunctional enzyme complexes that produce the bulk of fatty acids in the cell (45). FAS II occurs in mitochondria and chloroplasts (as well as in prokaryotes) and is catalyzed by the sequential action of individual enzymes (21, 51). The mitochondrial FAS II pathway produces octanoic acid, which is the precursor molecule for the synthesis of lipoic acid. Lipoic acid is the "swinging arm" cofactor that serves to shuttle reaction intermediates between the active sites of several mitochondrial keto acid dehydrogenase complexes (35). In yeast, lipoic acid is covalently bound to conserved lysine residues on three different proteins, the E2 subunit of pyruvate dehydrogenase (PDH) (34) and ␣-ketoglutarate dehydrogenase (␣-KDH) (38) and on the H protein of the glycine cleavage enzyme (GC) (33).Mitochondrial genes are expressed through a coordinated effort between the nuclear and the mitochondrial genomes. Mitochondrial DNA in eukaryotes encodes a small number of proteins that are components of the respiratory chain complexes and ATP synthase, as well as mitochondrial rRNAs and most tRNAs (1, 15). Other proteins required for mitochondrial function are encod...

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

“…6D). In addition, a marked decrease in mature RPM1, the RNA subunit of mitochondrial RNase P, accompanied by the accumulation of higher-molecular weight RNA species, was observed, in agreement with previous suggestions that endonucleolytic cleavages by tRNase Z contribute to the release of the mature RPM1 (Stribinskis et al 2001;Schonauer et al 2008). Also, the appearance of the 3 ′ -extended pre-RPM1-tRNA Pro * and the absence of pre-RPM1-tRNA Pro (detected by both RPM1 and tRNA Pro probes) in the mutant is consistent with a processing deficiency by Trz1.…”

“…However, the existence of the RPM1 # precursor lacking tRNA Pro (detected only with a probe specific for RPM1) argues that some 5 ′ tRNA processing by RNase P still takes place, even with a significantly reduced amount of mature RPM1. Therefore, accumulation of unprocessed pretRNA Pro is most likely a direct effect of Trz1 dysfunction, especially considering that similar RNA species are not generated in strains with defective RNase P (Stribinskis et al 1996(Stribinskis et al , 2001Schonauer et al 2008).…”

“…Probe names are indicated in parentheses, identities of RNA transcripts are shown on the right, graphic representations of corresponding mitochondrial transcription units as in Tzagoloff and Myers (1986); Foury et al (1998); Schonauer et al (2008), with the position of probes used for hybridization, are depicted on the left (C) or on the right (D). RNAs marked with diamonds represent aberrant RPM1 precursors or processing intermediates.…”

“…Analysis of the processing of the tRNA and its derivatives), were assigned based on their relative sizes, hybridization pattern, and previous reports (Stribinskis et al 1996(Stribinskis et al , 2001Schonauer et al 2008 (Fig. 6D).…”

Structure of human Fe–S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP–ISD11 interactions

“…Experimental evidence supports a role of ACP-associated fatty acids in the processing of mitochondrial RNA and expression of the respiratory complexes (14,70), synthesis of Fe-S cluster cofactors (1), and achieving the mature protein assembly and active conformation for the respiratory complexes (3,23). Thus, it appears from our results and the work of others that the evolutionarily conserved pathways of Fe-S cluster biosynthesis, oxidative phosphorylation, and mtFAS have been connected by LYR proteins and their respective acyl-ACP associations.…”