Abstract
In plants, post-transcriptional gene silencing (PTGS) represses gene expression by translation inhibition and cleavage of target mRNAs. The slicing activity is provided by argonaute 1 (AGO1), and the cleavage site is determined by sequence complementarity between the target mRNA and the microRNA (miRNA) or short interfering RNA (siRNA) loaded onto AGO1, to form the core of the RNA induced silencing complex (RISC). Following cleavage, the resulting 5′ fragment is modified at its 3′ end by the untemplated addition of uridines. Uridylation is proposed to facilitate RISC recycling and the degradation of the RISC 5′-cleavage fragment. Here, we detail a 3′ RACE-seq method to analyze the 3′ ends of 5′ fragments produced from RISC-cleaved transcripts. The protocol is based on the ligation of a primer at the 3′ end of RNA, followed by cDNA synthesis and the subsequent targeted amplification by PCR to generate amplicon libraries suitable for Illumina sequencing. A detailed data processing pipeline is provided to analyze nibbling and tailing at high resolution. Using this method, we compared the tailing and nibbling patterns of RISC-cleaved MYB33 and SPL13 transcripts between wild-type plants and mutant plants depleted for the terminal uridylyltransferases (TUTases) HESO1 and URT1. Our data reveal the respective contributions of HESO and URT1 in the uridylation of RISC-cleaved MYB33 and SPL13 transcripts, with HESO1 being the major TUTase involved in uridylating these fragments. Because of its depth, the 3′ RACE-seq method shows at high resolution that these RISC-generated 5′ RNA fragments are nibbled by a few nucleotides close to the cleavage site in the absence of uridylation. 3′ RACE-seq is a suitable approach for a reliable comparison of uridylation and nibbling patterns between mutants, a prerequisite to the identification of all factors involved in the clearance of RISC-generated 5′ mRNA fragments.
Highlights
Small RNAs are key regulators of gene expression (Borges and Martienssen, 2015; Bartel, 2018)
SiRNAs are generated from near-perfect double stranded RNAs or fully paired dsRNAs when the complementary strand is synthesized by a RNA-dependent RNA polymerase (RDR), which uses the sense strand as template. miRNAs and short interfering RNA (siRNA) are loaded onto members of the argonaute (AGO) protein family to form the core of RNA induced silencing complexes (RISCs) (Vaucheret, 2008; Zhang et al, 2015)
The base pairing of miRNAs with their targets is rather extensive, and mRNAs regulated by RISCs are repressed by argonaute 1 (AGO1)-mediated cleavage, and by translation repression (Chen, 2004; Brodersen et al, 2008; Yang et al, 2012; Li et al, 2013; Iwakawa and Tomari, 2015; Reis et al, 2015; Arribas-Hernández et al, 2016)
Summary
Small RNAs are key regulators of gene expression (Borges and Martienssen, 2015; Bartel, 2018). We detail a 3 RACE-seq method that has been optimized for analyzing the uridylation of 5 fragments from RISC-cleaved transcripts Those molecules are usually low abundant within the complex mixture of all cellular RNAs, and they exhibit a rather poor diversity, with a few untemplated nucleotides usually added at a precise RISC-mediated cleavage site. We illustrate the use of 3 RACE-seq to analyze the tailing and trimming patterns of MYB33 and SPL13 RISC 5 -cleavage fragments by comparing WT plants and mutants lacking HESO1 and URT1 This analysis revealed the respective contributions of both TUTases, and that the absence of uridylation results in the accumulation of 5 cleavage fragments nibbled by a few nucleotides close to the site cleaved by RISC
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