RNA uridylation and decay in plants

Abstract

RNA uridylation consists of the untemplated addition of uridines at the 3' extremity of an RNA molecule. RNA uridylation is catalysed by terminal uridylyltransferases (TUTases), which form a subgroup of the terminal nucleotidyltransferase family, to which poly(A) polymerases also belong. The key role of RNA uridylation is to regulate RNA degradation in a variety of eukaryotes, including fission yeast, plants and animals. In plants, RNA uridylation has been mostly studied in two model species, the green algae Chlamydomonas reinhardtii and the flowering plant Arabidopsis thaliana Plant TUTases target a variety of RNA substrates, differing in size and function. These RNA substrates include microRNAs (miRNAs), small interfering silencing RNAs (siRNAs), ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) and mRNA fragments generated during post-transcriptional gene silencing. Viral RNAs can also get uridylated during plant infection. We describe here the evolutionary history of plant TUTases and we summarize the diverse molecular functions of uridylation during RNA degradation processes in plants. We also outline key points of future research.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

Keywords: RNA decay; RNA degradation; mRNAs; terminal nucleotidyltransferase; uridylation.

Figure 1.

Domain organization of class I and class II TNTases of A. thaliana. The class I TNTase family is composed of 10 non-canonical poly(A) polymerases (ncPAPs) and four canonical poly(A) polymerases (cPAPs). The class II TNTase family contains the tRNA nucleotidyltransferase (tRNA-NT), also called the tRNA CCA-adding enzyme, and four bacterial PAP-like nucleotidyltransferases. Boxes represent conserved structural domains identified using the structural classification of proteins (SCOP) according to the superfamily database [25]. Non-conserved regions are drawn as lines. Each TNTase is identified by its AGI (Arabidopsis Genome Initiative) reference gene model. The numbers of all gene models are shown in parentheses. Finally, names are shown for the 10 TNTases that have been studied to date. The numbers on the right indicate the number of amino acids for each TNTase. The colour code for the Superfamily SCOP domains is indicated on the figure. The vertical black bar drawn in the nucleotidyltransferase domain of four of the five class II TNTases represents a motif discriminating bacterial PAP-like nucleotidyltransferases from bacterial tRNA-NT [15].

Figure 2.

Phylogenetic relationship among A. thaliana class I ncPAPs and four human ncPAPs. The nucleotidyltransferase domains SCOP 81301 and the PAP/OAS1 substrate-binding domains SCOP 81631 of the 10 class I ncPAPs of Arabidopsis and four human ncPAPs were aligned with Muscle (v. 3.8.31) [37]. The maximum-likelihood tree was generated using PhyML (v. 3.1) on Phylogeny.fr [38] and edited using FigTree (v. 1.4.3, http://tree.bio.ed.ac.uk/software/figtree/). Arabidopsis and human ncPAPs are indicated in regular and italic characters, respectively. Support values (approximate likelihood-ratio statistical test, aLRT v 3.0) are shown on branches. The scale bar represents the number of substitutions per amino acid site. HESO1, URT1 and two other TNTases form a cluster with human TUT7 and TUT4. MEE44 and TRL form a cluster with human TENT4A and TENT4B. The remaining four TNTases form a separated cluster. HESO1, URT1 and TRL, the three class I ncPAPs that have been functionally characterized in Arabidopsis, are indicated in bold.

Figure 3.

Copy number of HESO1 and URT1 in 79 representative species of Archaeplastida. The phylogenic relationship between the 79 species analysed in this study was visualized using Phylostatic [44]. Full species names are grouped by taxonomic clades indicated on the right. The colour code for URT1 and HESO1 in the different clades is conserved in figures 4 and 5. The numbers of copies detected for HESO1 and URT1 are indicated for each species. Sequences in FASTA format are given in electronic supplementary material, Datasets S1 and S2 for HESO1 and URT1, respectively.

Figure 4.

Phylogenetic relationship between URT1 and HESO1 sequences among 79 representative species of Archaeplastida. The phylogenetic tree was generated using the maximum-likelihood method and WAG substitution model implemented in PhyML (v. 3.1) [45,46]; see electronic supplementary material, Dataset S3 for sequence alignment). The tree was edited using iTOL (v. 4.2.1) [47]. Colour code for taxonomic clades is defined in figure 3. Statistical values for the first branches (approximate likelihood-ratio test, aLRT v. 3.0) support that URT1 and HESO1 proteins form two distinct clades. The scale bar represents the number of substitutions per amino acid site.

Figure 5.

Phylogenetic relationship of URT1 and HESO1 isoforms among 11 species of Poales. Sequences of HESO1 and URT1 isoforms from 10 Poaceae (Po) and 1 Bromeliaceae (Br) were aligned separately. Full names of species are given in figure 3. The analysis was performed as described for figure 4 except that the trees were edited with FigTree (v. 1.4.3, http://tree.bio.ed.ac.uk/software/figtree/). Support values (approximate likelihood-ratio statistical test, aLRT v. 3.0) are shown on branches. The sequence alignments for HESO1 and URT1 used to build the trees are given in electronic supplementary material, Datasets S4 and S5, respectively.

Figure 6.

Expression of URT1 and HESO1 isoforms from selected species of the Poaceae family. The diagram was drawn based on the transcriptomic data deposited in Phytozome v. 12.1 (https://phytozome.jgi.doe.gov) [49], in Next-Gen Sequence DBs (https://mpss.danforthcenter.org/dbs) [50], in eFP browser (http://bar.utoronto.ca) [51] and from the RNA-seq data from [52]. Expression data were analysed for Poales species from the BOP clade (B. distachyon, O. sativa) and the PACMAD clade (P. hallii, S. italica, S. bicolor, Z. mays) of Poaceae and for a Bromeliaceae (A. comosus).

Figure 7.

Small RNA uridylation and decay. 2′-O-methylation deposited by the methyltransferase HEN1 protects against uridylation by HESO1 or by URT1. The exoribonucleases' SDNs can nibble methylated sRNAs that are loaded in AGO, thereby generating nibbled, unprotected sRNAs. These unprotected siRNAs or miRNAs are targeted by HESO1 (or URT1 for a subset of miRNAs). The uridylated small RNAs are subsequently degraded by a 3′ to 5′ exoribonucleolytic activity(ies) that is unknown in Arabidopsis and was proposed to be RRP6 in C. reinhardtii [42].

Figure 8.

Uridylation by HESO1 promotes degradation of 5′ fragments of RISC-cleaved mRNAs. RISC cleavage of mRNAs generates a 3′ cleavage fragment that is degraded by XRN4, and a 5′ cleavage fragment. The 5′ cleavage fragment is uridylated by HESO1, which binds RISC, but can also be decapped by the DCP1/2 complex. The exoribonucleases RICE1/2, which are recruited by RISC, nibble the uridylated 5′ cleavage fragment. This nibbling helps RISC dissociation and RISC recycling. Finally, the 5′ cleavage fragment is degraded by XRN4 and the RNA exosome.