| Abstract [eng] |
Animal Hen1 methyltransferases are composed of the N-terminal methyltransferase domain and the variable-length C-terminal region, and catalyze the S-adenosyl-L-methionine (AdoMet) dependent methylation of the 2'-O-ribose of the 3'-terminal nucleotide in a subset of small non-coding RNAs. This modification protects small RNAs from degradation, meanwhile the deficiency of the Hen1 protein causes detrimental phenotypic changes to particular organisms. Current work presents the first detailed characterization of the enzymatic reactions catalysed by animal Hen1 methyltransferases: we have discovered that the enzymatic activity of Drosophila melanogaster DmHen1 and Homo sapiens HsHen1 depends on Co2+ and Co3+ ions; we have found that in vitro these proteins can transfer not only methyl group, but also larger side chains from synthetic AdoMet analogues on both natural 22-28 nt and longer RNA substrates; we have shown that the C-terminal region of DmHen1 is unnecessary and even inhibits the enzymatic activity of the protein. In the current study we have applied DmHen1 and its variant without the C-terminal sequence, DmHen1ΔC, for the one-step and two-step labeling of single-stranded RNA and its subsequent visualization, enrichment, and synthesis of copy DNA (cDNA). Based on the latter reaction, we have developed the mDOT-seq (methyltransferase-Directed Orthogonal Tagging and RNA sequencing), the method for the cDNA library preparation for single-stranded RNA sequencing. With mDOT-seq in hand we have not only demonstrated that it can be applied for the sequencing of small eukaryotic RNAs (such as microRNA or siRNA), but also for the first time characterized the 50-500 nt small regulatory RNAs from probiotic Lactobacillus casei BL23. |
