Biochemical and structural insights into the function of small RNA methyltransferase Hen1 in animals. Mus musculus

HUA ENHANCER 1 (Hen1) is a RNA 2'-O-methyltransferase that modifies small non-coding RNAs (sncRNAs) by adding a methyl group to the 2'-OH of 3'-terminal nucleotides, thereby protecting them from other modifications and degradation. The molecular mechanism underlying the methylation of small RNA duplexes has been revealed by the crystal structure of Hen1 in Arabidopsis (AtHen1). However, the molecular basis of single-stranded RNA methylation by mammalian Hen1 homologues, especially p-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), remains elusive. Here we show that mouse Hen1 (mHen1) is responsive for the 3’ end methylation of classical piRNAs, as well as for the non-canonical piRNAs derived from ribosomal RNA (rRNAs), small nuclear RNAs (snRNAs) and transfer RNAs (tRNAs) in mouse spermatogonial stem cells (SSCs). Moreover, we identified a class of transfer RNA (tRNA)-derived sncRNAs as novel substrates of mHen1, which we refer to as Hen1-methylated tRNA-derived small RNAs. We determined the crystal structure of the catalytic domain of human Hen1 (HsHen1) in complex with its cofactor AdoMet at 1.8 Å resolution. Comparisons of the structures of HsHen1 and AtHen1 reveal a similar active site for binding a divalent cation and AdoMet. An in vitro methyltransferase assay indicated that the complete catalytic domain of HsHen1 is sufficient to methylate a specific length of single-stranded RNAs in a manganese-dependent manner. In conclusion, our functional and structural findings provide important insights into the methylation details of sncRNAs in mouse SSCs, and the catalytic mechanism of mammalian Hen1 Overall design: Small RNA profiling of wild-type, mhen1-/- and mili-/- SSC, and which small RNAs have 3' modification by treatment with NaIO4.