Isolation and RNA-binding analysis of NAD + -isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe

Abstract

Krebs cycle NAD+ -isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiae COX2 leaders. In contrast. Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA.

Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins

“…Using the ''reactome '' annotation (Joshi-Tope et al, 2005), the HeLa mRNA interactome harbors 17 enzymes of intermediary metabolism, and the extended class IV list increases this count to 46 (Table 1). In part, this list confirms earlier experiments (Cie sla, 2006;Elzinga et al, 1993Elzinga et al, , 2000Kiri and Goldspink, 2002;Liu et al, 2001;Nagy and Rigby, 1995;Nakagawa et al, 1995;Pioli et al, 2002;Shetty et al, 2004), and it also identifies metabolic enzymes not previously known as RBPs. We validated four of these as RBPs by the dual fluorescence assay (Figure 3C); ENO1 and SHMT2 were also validated by sequencing of associated RNAs (Figures 3D and 3E).…”