Dehydrogenases from All Three Domains of Life Cleave RNA

Abstract

Specific interactions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with RNA have been reported both in vitro and in vivo. We show that eukaryotic and bacterial GAPDH and two proteins from the hyperthermophilic archaeon Sulfolobus solfataricus, which are annotated as dehydrogenases, cleave RNA producing similar degradation patterns. RNA cleavage is most efficient at 60°C, at MgCl 2 concentrations up to 5 mM, and takes place between pyrimidine and adenosine. The RNase active center of the putative aspartate semialdehyde dehydrogenase from S. solfataricus is located within the N-terminal 73 amino acids, which comprise the first mononucleotide-binding site of the predicted Rossmann fold. Thus, RNA cleavage has to be taken into account in the ongoing discussion of the possible biological function of RNA binding by dehydrogenases.It is known that dehydrogenases and other metabolic enzymes can bind RNA. In numerous studies on RNA binding, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1 was identified as the major RNA-binding protein. It was shown that human and/or rabbit muscle GAPDH selectively binds tRNA (1), AU-rich sequences at the 3Ј-untranslated region conferring instability of the corresponding mRNAs (2), hammerhead ribozyme (3), and viral cis-acting regulatory RNA elements (4 -7). The selectivity of GAPDH binding to RNA was demonstrated in vitro and in vivo (4,8,9). Furthermore, one of the proteins that specifically binds to a small stable RNA from Mycoplasma capricolum was identified as bacterial GAPDH (10). On the other hand, human GAPDH was also identified as a major protein that binds to single-stranded DNA (ssDNA) and oligodeoxynucleotides containing the TAAAT motif (11,12).Recently it was proposed that the NAD-binding structure (Rossmann fold) of GAPDH represents a novel RNA binding domain that provides a molecular basis for RNA recognition by dinucleotide-binding metabolic enzymes (13). Baker et al. (14) provided another line of evidence for the relationship between dehydrogenases and enzymes interacting with RNA. They revealed that spinach CSP41, an mRNA-binding protein and ribonuclease (15), is homologous to nucleotide-sugar epimerases and hydroxysteroid dehydrogenases and proposed that these proteins share a common ancestor.We are interested in the identification of archaeal endoribonucleases and therefore monitored protein fractions from the hyperthermophilic archaeon Sulfolobus solfataricus using RNase activity assays. Two major proteins were copurified in a cell fraction with RNase activity. Surprisingly, they were identified as hypothetical dehydrogenases. During their biochemical characterization as endonucleases, we found that eukaryotic and bacterial GAPDH enzymes also possess RNase activity. Our results show for the first time that the key glycolytic enzyme GAPDH, which possesses RNA-binding capability, also acts as RNase. The finding that two archaeal proteins annotated as dehydrogenases, as well as eukaryotic and bacterial GAPDH, efficiently cleave RNA suggests that dehydrogena...

Malonyl-Coenzyme A Reductase in the Modified 3-Hydroxypropionate Cycle for Autotrophic Carbon Fixation in Archaeal Metallosphaera and Sulfolobus spp

“…2C). Aberrant migration in SDS-PAGE was also reported for aspartate-semialdehyde dehydrogenase from Sulfolobus solfataricus (11). Gel filtration chromatography showed a native molecular mass of 160 kDa, which would suggest a homotetrameric subunit composition (however, see below).…”

“…This altered conformation for cofactor binding at the N terminus of the enzyme may allow the binding of RNA and/or CoA in the case of malonyl-CoA reductase. For aspartatesemialdehyde dehydrogenase from S. solfataricus it has been shown that RNA binds to the same region of the enzyme as NADPH (11). However, sequence comparison does not identify a structural feature responsible for the use of a CoAactivated acid substrate in the case of malonyl-CoA reductase versus a phosphate-activated acid substrate in the case of aspartate-semialdehyde dehydrogenase.…”

“…The RNA size varies from 60 to 180 nucleotides, which could be interpreted as a hint for nonspecific cleaving of RNA. RNA cleavage has been shown for other metabolic enzymes, e.g., glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, phosphoglycerate kinase, glucose 6-phosphate dehydrogenase, and glutamate dehydrogenase; however, the physiological role is not known (8,11) Testing to determine whether malonyl-CoA reductase cleaves RNA or RNA affects malonyl-CoA reductase activity, either positively or negatively, has not been done. Thus, an answer to the question of whether the RNA binding and possibly RNA cleavage by malonyl-CoA reductase plays a physiological role requires further detailed investigation and was out of the scope of this work.…”

Species-Specific Differences in Translational Regulation of Dihydrofolate Reductase

“…A number of dehydrogenases have been shown to interact with RNA through their NAD/NADPH binding sites (Hentze, 1994;Nagy et al, 2000). One of the most noteworthy, glyceraldehyde-3-phosphate dehydrogenase, binds tRNA, adenylate uridylate-rich sequences at the 3Ј-untranslated region of mRNA, hammerhead ribozyme, and viral cis-acting regulatory elements (Evguenieva-Hackenberg et al, 2002). The possibility that human DHFR may bind to other mRNA species is currently being explored.…”

Purification, identification and characterization of mammalian endoribonucleases that degrade c-myc mRNA in vitro.

“…GAPDH has also been shown to bind single-stranded DNA containing a TAAAT motif. In fact, several dehydrogenase enzymes from multiple domains of life have been shown to possess RNA-and DNA-binding capabilities (Ciesla 2006;Evguenieva-Hackenberg et al 2002). In light of this it would be of interest to test HADHSC's ability to bind both double-and single-stranded DNA.…”