Identification of substrates of the small RNA methyltransferase Hen1 in mouse spermatogonial stem cells and analysis of its methyl-transfer domain

Abstract

Small noncoding RNAs (sncRNAs) regulate many genes in eukaryotic cells. Hua enhancer 1 (Hen1) is a 2'--methyltransferase that adds a methyl group to the 2'-OH of the 3'-terminal nucleotide of sncRNAs. The types and properties of sncRNAs may vary among different species, and the domain composition, structure, and function of Hen1 proteins differ accordingly. In mammals, Hen1 specifically methylates sncRNAs called P-element-induced wimpy testis-interacting RNAs (piRNAs). However, other types of sncRNAs that are methylated by Hen1 have not yet been reported, and the structures and the substrates of mammalian Hen1 remain unknown. Here, we report that mouse Hen1 (mHen1) performs 3'-end methylation of classical piRNAs, as well as those of most noncanonical piRNAs derived from rRNAs, small nuclear RNAs and tRNAs in murine spermatogonial stem cells. Moreover, we found that a distinct class of tRNA-derived sncRNAs are mHen1 substrates. We further determined the crystal structure of the putative methyltransferase domain of human Hen1 (HsHen1) in complex with its cofactor AdoMet at 2.0 Å resolution. We observed that HsHen1 has an active site similar to that of plant Hen1. We further found that the putative catalytic domain of HsHen1 alone exhibits no activity. However, an FPP motif at its N terminus conferred full activity to this domain, and additional binding assays suggested that the FPP motif is important for substrate binding. Our findings shed light on its methylation substrates in mouse spermatogonial stem cells and the substrate-recognition mechanism of mammalian Hen1.

RNA ribose methylation (2′-O-methylation): Occurrence, biosynthesis and biological functions

“…In contrast to plants, HEN1 homologs in animals act on single-stranded RNAs such as germline piRNAs and Ago2-associated siRNAs (but not Ago1-associated miRNA) as well as tRNA-derived sncRNAs [147] to deposit a 2'-O-methyl group on their 3' termini [147,148]. Interestingly, the substrates of HEN1 are quite different between plants and other species and this was recently proposed to be due to a conserved FxPP motif that is essential for substrate recognition [147].…”

“…In Arabidopsis, 2'-O-methylation at the 3'-end of RNA is performed by the RNA methyltransferase Hua Enhancer 1 (HEN1), which methylates both miRNA/miRNA and siRNA/siRNA duplexes with a preference for 21-24 nt RNA duplexes with a 2 nt overhangs [145,146]. In contrast to plants, HEN1 homologs in animals act on single-stranded RNAs such as germline piRNAs and Ago2-associated siRNAs (but not Ago1-associated miRNA) as well as tRNA-derived sncRNAs [147] to deposit a 2'-O-methyl group on their 3' termini [147,148]. Interestingly, the substrates of HEN1 are quite different between plants and other species and this was recently proposed to be due to a conserved FxPP motif that is essential for substrate recognition [147].…”

Investigating the Viral Suppressor HC-Pro Inhibiting Small RNA Methylation through Functional Comparison of HEN1 in Angiosperm and Bryophyte

“…The AtHEN1 and MpHEN1 contain two dsRBD domains that can specifically bind to an miRNA/miRNA* or siRNAs duplex but not single-stranded miRNAs or siRNAs. In contrast to plants, the HEN1 orthologs in animals can act on piRNAs or single-stranded siRNAs [40]. Moreover, not all small RNAs in animals are methylated by HEN1 orthologs.…”

“…The alignment data indicated that some SAM-binding residues and metal-binding residues were highly conserved in the MTase domain (Figure 9). The FXPP motif in the N terminus of the MTase domain is essential for substrate recognition of eukaryotic HEN1 [40]. The alignment demonstrated that plant HEN1s displayed an FXP(P/S/C) sequence in the corresponding position (Figure 9).…”

Knockout Gene-Based Evidence for PIWI-Interacting RNA Pathway in Mammals

“…The exonuclease trims the 3 -end of piRNA intermediates to their mature length (Ding et al, 2017;Zhang et al, 2017b;Bronkhorst and Ketting, 2018;Nishimura et al, 2018). The 3 -end of mature piRNA is 2 -O-methylated by HENMT1, yet correct 3 truncation is not necessary for 3 -end 2 -O-methylation (Yang et al, 2006;Zhai and Meyers, 2012;Peng et al, 2018). In addition, TUT4/7 mediates the 3 uridylation of 30-to 31-nt-long piRNAs, but its effect is unknown (Morgan et al, 2019).…”