Uridylation of miRNAs by HEN1 SUPPRESSOR1 in Arabidopsis

Abstract

Summary HEN1-mediated 2′-O-methylation has been shown to be a key mechanism to protect plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) as well as animal piwi-interacting RNAs (piRNAs) from degradation and 3′ terminal uridylation [1–8]. However, enzymes uridylating unmethylated miRNAs, siRNAs, or piRNAs in hen1 are unknown. In this study, a genetic screen identified a second-site mutation hen1 suppressor1-2 (heso1-2) that partially suppresses the morphological phenotypes of the hypomorphic hen1-2 allele and the null hen1-1 allele in Arabidopsis. HESO1 encodes a terminal nucleotidyl transferase that prefers to add untemplated uridine to the 3′ end of RNA, which is completely abolished by 2′-O-methylation. heso1-2 affects the profile of u-tailed miRNAs and siRNAs and increases the abundance of truncated and/or normal sized ones in hen1, which often results in increased total amount of miRNAs and siRNAs in hen1. In contrast, overexpressing HESO1 in hen1-2 causes more severe morphological defects and less accumulation of miRNAs. These results demonstrate that HESO1 is an enzyme uridylating unmethylated miRNAs and siRNAs in hen1. These observations also suggest that uridylation may destabilize unmethylated miRNAs through an unknown mechanism and compete with 3′-to-5′ exoribonuclease activities in hen1. This study shall have implications on piRNA uridylation in hen1 in animals.

Plant MicroRNAs Display Differential 3' Truncation and Tailing Modifications That Are ARGONAUTE1 Dependent and Conserved Across Species

“…The cross-species conservation of 39 modifications of miRNAs suggests that it is the sequence of the mature miRNA that is the primary determinant of these modifications, both in the wild type and the hen1 mutants. Loss-of-function mutations in the genes that catalyze truncation (possibly via SDN1 and 2) and uridylation (HESO1) have a clear impact on miRNA stability and accumulation (Ramachandran and Chen, 2008;Ren et al, 2012;Zhao et al, 2012b). Therefore, knowing that miRNAs vary substantially in numerous aspects of their biogenesis (e.g., expression, structure, processing, etc.…”

“…Even in an Arabidopsis wild-type background with functional HEN1, miR158 was both truncated and tailed at substantial levels, levels somewhat similar to this miRNA in the hen1 mutants ( Figure 2A). HEN1 SUPPRESSOR1 (HESO1) is a nucleotidyltransferase that can uridylate unmethylated miRNAs and promote their degradation (Ren et al, 2012;Zhao et al, 2012b). Arabidopsis HESO1 was identified because its loss of function can suppress the phenotype of hen1-8 by reducing levels of uridylation and increasing miRNA accumulation in general.…”

“…Comparisons performed by cloning and sequencing individual miRNAs in wild-type and hen1-1 backgrounds showed the addition of predominantly uridine (U) nucleotides at the 39 end of some miRNAs in a hen1-1 mutant . The U-rich tail of unmethylated miRNAs in Arabidopsis hen1 mutants is added by nucleotidyl transferase HEN1 SUPPRESSOR1 (HESO1), whose mutation can partially rescue the phenotype of hen1 mutants by increasing miRNA accumulation (Ren et al, 2012;Zhao et al, 2012b). A family of SMALL RNA DEGRADING NUCLEASE (SDN) proteins was identified in Arabidopsis that degrades mature miRNAs (Ramachandran and Chen, 2008).…”

Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage

“…In contrast, uridylation of some siRNAs in Caenorhabditis elegans restricts them to CSR-1 (an AGO protein) and reduces their abundance, which is required for proper chromosome segregation (18). In the green algae Chlamydomonas reinhardtii and the flowering plant Arabidopsis, uridylation causes the degradation of miRNAs and siRNAs (19)(20)(21). Enzymes that uridylate miRNAs and siRNAs have been identified in both animals and plants.…”

Deep Sequencing from hen1 Mutants to Identify Small RNA 3' Modifications

“…However, in vitro analysis of the SDNs suggests that they degrade miRNAs 3 0 to 5 0 , leaving a small fragment of 8 or 9 nucleotides in length, and this is quite distinct from the typical 1-or 2-nucleotide shorter size observed for truncated miRNAs in RNA gel blots from hen1 mutants Yu et al 2005). More recent work from Chen and Yu laboratories identified a nucleotidyl transferase HEN1 SUPPRESSOR1 (HESO1) that is responsible for the addition of the uridine-rich tail to the end of the miRNAs, which are not protected by methylation (Ren et al 2012;Zhao et al 2012). Other genes in the same family, which include poly(U) polymerases, were previously shown in animals to modify and regulate the stability of the pre-miRNA (cleaved hairpin) of let-7 (Lehrbach et al 2009).…”