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

On Apr. 27^th, J Biol Chem published online a work entitled “Identification of substrates of the small RNA methyltransferase Hen1 in mouse spermatogonial stem cells and analysis of its methyl-transfer domain” from the research teams of Dr. HUANG Ying and Dr. WU Ligang from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (SIBCB, CAS). This work identified a novel class of small noncoding RNAs derived from tRNAs is Hen1 substrate in mouse SSCs. We also found that a FxPP motif at the N-terminus of the methyltransfer domain is important for Hen1 activity.

Small noncoding RNAs are often methylated at their 3¢-end, such as miRNAs and siRNAs in plant and piRNAs in animals. The methylation is catalyzed by a methyltransferase protein HUA ENHANCER 1 (Hen1). The previously reported Arabidopsis Hen1 crystal structure revealed that multiple domains in plant Hen1 cooperate to bind miRNA/miRNA* duplex and mediate the methyl transfer reaction in a magnesium dependent manner.

However, animal Hen1 could catalyze the methylation of piRNAs and single-stranded siRNAs, but not small RNA duplexes. The domain architecture, the length of the protein and the substrate of animal Hen1 is quite different from those of plant Hen1.

In this work, they systematically studied the methylation substrate of Hen1 in mouse SSCs by sodium periodate-assisted deep sequencing. Besides the canonical piRNAs, they identified a class of tRNA-derived small noncoding RNAs (hmtsRNAs) that is 31–33 nt in length as a novel substrate of mouse Hen1. These hmtsRNAs are abundant in mature sperm, suggesting that the newly identified hmtsRNAs may function in latter stages of spermatogenesis and embryonic development. The crystal structure of the catalytic domain of human Hen1 was also studied. An FxPP motif at the N-terminus of the catalytic domain that is conserved in eukaryotic Hen1 proteins is essential for Hen1 substrate recognition and catalytic activity. The structural and functional studies provide mechanistic insights into the role of mouse and Hen1 in methylation of piRNAs and other small noncoding RNAs in mammals.

PENG Ling, ZHANG Fengjuan, and SHANG Renfu are the first authors of this work. This study was supported by National Natural Science Foundation of China, Strategic Priority Research Program of the Chinese Academic of Sciences, Chinese Academy of Sciences Facility-based Open Research Program and the State Key Laboratory of Molecular Biology.

Fig. 1. (A) Strategies to profile the methylated pattern of sncRNAs in mouse SSCs. (B) Overall structure of the crystal structure of HsHen1 MTase domain in complex with SAM.