Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

Abstract

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

HASTY , the Arabidopsis EXPORTIN5 ortholog, regulates cell‐to‐cell and vascular microRNA movement

“…To explore the possibility that elevated miRNA tailing/trimming/misprocessing might occur in hst , we mined the recent hst‐15 versus WT sRNA sequencing data from Cambiagno et al ( 2021 ). Tailing/trimming was assessed with a method similar to that described recently in Giudicatti et al ( 2021 ), for which publicly available sRNA sequencing data for the hen1 and hen1 heso1 mutants (Wang et al , 2018 ) were also used in parallel. While tailing/trimming was readily detected, as reported, in the latter two mutants, it was not observed in hst‐15 (Appendix Fig S10B ) and nor was miRNA misprocessing, as assessed by testing the accumulation of 20–22‐nt‐long miRNA isoforms deviating by up to +/− 5 nucleotides from their cognate, annotated sequences (Iki et al , 2018 ) (Appendix Fig S10B ).…”

“…Trimming, tailing, and isoform estimates (Appendix Fig S10B ) were calculated with a method inspired from Giudicatti et al ( 2021 ), which identifies potentially unaltered, trimmed, or tailed miRNAs, and assigns an index by dividing the number of trimmed or tailed molecules with the one of the annotated (unaltered) miRNA sequences. Raw sequencing data of hst‐15 , hen1 , and hen1 heso1 were obtained from SRA (accessions ERP126434 and SRX3405447).…”

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation

“…Stability of miRNAs is assured not only by 3′ ends 2′- O -methylation but also by its binding to AGO1. Strong hypomorphic mutants of AGO1 show reduced a level of mature miRNAs, while in weak ago1 alleles, protection of miRNAs from trimming or uridylation could be observed [ 66 , 135 ]. Slicing or recognition of the target mRNA using RISC triggers miRNA trimming or tailing, allowing for miRNA degradation and AGO1 release [ 135 ].…”

“…Strong hypomorphic mutants of AGO1 show reduced a level of mature miRNAs, while in weak ago1 alleles, protection of miRNAs from trimming or uridylation could be observed [ 66 , 135 ]. Slicing or recognition of the target mRNA using RISC triggers miRNA trimming or tailing, allowing for miRNA degradation and AGO1 release [ 135 ]. Moreover, extended interaction of RISC with target mRNA results in AGO1 and miRNA degradation [ 135 , 136 ].…”

“…Slicing or recognition of the target mRNA using RISC triggers miRNA trimming or tailing, allowing for miRNA degradation and AGO1 release [ 135 ]. Moreover, extended interaction of RISC with target mRNA results in AGO1 and miRNA degradation [ 135 , 136 ]. It was shown that AGO1 directly interacts with HESO1, URT1 and ATRM2, further indicating the active role of AGO1 in miRNA turnover [ 131 , 137 , 138 ].…”