Allosteric Inhibition of NAD⁺-Specific Isocitrate Dehydrogenase by a Mitochondrial mRNA

Abstract

NAD+-specific isocitrate dehydrogenase (IDH) has been reported to bind sequences in 5'-untranslated regions of yeast mitochondrial mRNAs. In the current study, an RNA transcript containing the 5'-untranslated region of the mRNA from the yeast mitochondrial COX2 gene is shown to be an allosteric inhibitor of the affinity-purified yeast enzyme. At 0.1 microM concentrations of the transcript, velocity of the IDH reaction is reduced to 20% of the value obtained in the absence of the RNA transcript. This inhibition is due to a 2. 5-fold increase in the S0.5 value for isocitrate. Significant inhibition of IDH activity is also obtained with a transcript containing a portion of the 5'-untranslated region of the yeast mitochondrial ATP9 gene and with an antisense form of the COX2 transcript, both of which contain potential stem-loop secondary structures implicated in binding of IDH. In contrast, much higher concentrations of yeast tRNA or poly(A)mRNA, respectively, 33- and 60-fold greater than that required for the COX2 transcript, are required to produce a 50% decrease in velocity. These results suggest that inhibition of activity is relatively specific for the 5'-untranslated regions of mitochondrial mRNAs. All measurable inhibition of IDH activity by RNA is eliminated by addition of 100 microM concentrations of the allosteric activator AMP. At equivalent concentrations, dAMP is less efficient than AMP as an allosteric activator of IDH and is proportionally less effective in protecting against inhibition of activity by the COX2 transcript. Other nucleotides that are not allosteric activators fail to protect IDH activity from inhibitory effects of RNA. Thus, alleviation of catalytic inhibition of IDH by mitochondrial mRNA correlates with the property of allosteric activation.

Complementary Profiling of Gene Expression at the Transcriptome and Proteome Levels in Saccharomyces cerevisiae

“…The branch point of these two cycles occurs at the protein Idh1p (41), which showed no significant change in abundance on either the mRNA or protein levels. This is consistent with recently published evidence that Idh1p is regulated allosterically by mitochondrial mRNA species (42), and therefore no change in abundance of the protein on either carbon source may be expected. The allosteric inactivation of Idh1p channels the metabolic flow to its competitor Icl1p and therefore into the glyoxylate cycle in what is known as the glyoxylate bypass (41).…”

Global Transcription Analysis of Krebs Tricarboxylic Acid Cycle Mutants Reveals an Alternating Pattern of Gene Expression and Effects on Hypoxic and Oxidative Genes

“…These functions may not be distinct, because mRNA binding by isocitrate dehydrogenase inhibits its catalytic activity (Anderson and McAlister-Henn, 2000). These observations suggest a mechanism whereby TCA cycle metabolic flux and the synthesis of respiratory complexes are coordinately regulated (Anderson and McAlister-Henn, 2000). Mutations in CIT1 also suppress the mtDNA instability of isocitrate dehydrogenase dysfunctional cells (our unpublished results), indicating that these two properties are functionally linked.…”

“…However, it is not clear whether the mtDNA instability associated with isocitrate dehydrogenase dysfunction results from the loss of catalytic activity or from the aberrant expression and turnover of mitochondrial respiratory complexes (de Jong et al, 2000;Lin et al, 2001). These functions may not be distinct, because mRNA binding by isocitrate dehydrogenase inhibits its catalytic activity (Anderson and McAlister-Henn, 2000). These observations suggest a mechanism whereby TCA cycle metabolic flux and the synthesis of respiratory complexes are coordinately regulated (Anderson and McAlister-Henn, 2000).…”

“…Yeast isocitrate dehydrogenase binds to mitochondrially encoded mRNAs and appears to regulate their translation (Elzinga et al, 1993). RNA binding also affects isocitrate dehydrogenase catalytic activity, suggesting a mechanism for coordinating expression of mitochondrial respiratory complexes and TCA cycle oxidative metabolism (Anderson et al, 2000).…”

RNA templating the epigenome

“…IDH binds to the 5'-end of mitochondrial mRNAs, and this binding inhibits its activity. 53,54 Newly developed whole-transcriptome approaches to identifying RNA-protein interactions will surely aid in identifying new RNAbinding enzymes and potential lincRNAenzyme complexes. [55][56][57] LincRNAs such as HOTAIR and Xist bind directly to chromatin remodeling enzyme complexes; whether or not they directly modulate their activity remains to be discovered.…”