Poster abstracts

Poster number 49 submitted by Kent Redman

Expression and preliminary studies of YNL022p, a putative rRNA methyltransferase.

Kent L. Redman (Indiana University School of Medicine-Fort Wayne), Jennifer L. Craft (Department of Biology, Ball State University)

Abstract:
Posttranscriptional modifications in Saccharomyces cerevisiae produce 5-methylcytosine (m⁵C) in several tRNAs and in 25S rRNA. Trm4p methylates tRNA, so either or both of the putative RNA m⁵C methyltransferases encoded by the yeast genome (Nop2p and YNL022p) could be responsible for rRNA methylation. To initiate studies of YNL022p, the corresponding open reading frame was amplified and inserted into the pET28b vector so that the expressed protein includes a His₆ tag. Only insoluble protein was formed after induction of BL21(DE3) cells at 37 ^oC. Protein solubility was maximal, yet poor when YNL022c was expressed at 30 ^oC. An adequate level of YNL022c expression for analysis was achieved by coexpression of the His₆ tagged construct with bacterial chaperone proteins at 30 ^oC.
Assays measuring the incorporation of radiolabel from ³H-[methyl]-AdoMet into total RNA revealed greater incorporation of isotope into RNA with extracts from YNL022p expressing cells than with control extracts. However, only marginally more radiolabel was introduced into methyl-deficient RNA isolated from yeast lacking YNL022c than was incorporated into methyl-replete RNA. To characterize methylated RNAs more specifically, assay mixtures were analyzed by SDS-PAGE. Control and YNL022p extracts methylated tRNA-sized molecules, but only the YNL022p extract generated a large methylated product. Further specificity was indicated by the production of the large product only with methyl-deficient RNA. Since large RNAs are not well resolved by SDS-PAGE, methylated RNAs were separated in denaturing agarose gels. Like the previous experiments, only YNL022p preparations were found to cause the methylation of a large RNA and the radiolabeled RNA comigrated with the 25S rRNA marker. Therefore, YNL022c appears to be a rRNA m⁵C methyltransferase. When partially purified rRNA is used as the substrate, a small RNA molecule is highly methylated by the YNL022p extract indicating the possibility of an additional substrate for the enzyme. Further analysis of YNL022p is in progress.

Keywords: methylation, 5-methylcytosine, RNA