Extensive profiling in Arabidopsis reveals abundant polysome-associated 24-nt small RNAs including AGO5-dependent pseudogene-derived siRNAs

Abstract

In a reductionist perspective, plant silencing small (s)RNAs are often classified as mediating nuclear transcriptional gene silencing (TGS) or cytosolic posttranscriptional gene silencing (PTGS). Among the PTGS diagnostics is the association of AGOs and their sRNA cargos with the translation apparatus. In Arabidopsis, this is observed for AGO1 loaded with micro(mi)RNAs and, accordingly, translational-repression (TR) is one layer of plant miRNA action. Using AGO1:miRNA-mediated TR as a paradigm, we explored, with two unrelated polysome-isolation methods, which, among the ten Arabidopsis AGOs and numerous sRNA classes, interact with translation. We found that representatives of all three AGO-clades associate with polysomes, including the TGS-effector AGO4 and stereotypical 24-nt sRNAs that normally mediate TGS of transposons/repeats. Strikingly, approximately half of these annotated 24-nt siRNAs displayed unique matches in coding regions/introns of genes, and in pseudogenes, but not in transposons/repeats commonly found in their vicinity. Protein-coding gene-derived 24-nt sRNAs correlate with gene-body methylation. Those derived from pseudogenes belong to two main clusters defined by their parental-gene expression patterns, and are vastly enriched in AGO5, itself found on polysomes. Based on their tight expression pattern in developing and mature siliques, their biogenesis, and genomic/epigenomic features of their loci-of-origin, we discuss potential roles for these hitherto unknown polysome-enriched, pseudogene-derived siRNAs.

Keywords: eukaryota; plants; polysome; small RNAs.

FIGURE 1.

An AGO1 miRNA-RISC associates with ribosomal complexes in Arabidopsis. (A) Immunoprecipitation of ribosomal complexes from inflorescences via constitutive Flag-tagged RPL18 in two independent experiments (left and right panel); see main text for details. The abundance of AGO1 and selected miRNAs in the various fractions were measured by western and northern analysis using an antibody against native AGO1 and labeled complementary oligonucleotide probes, respectively. (FT) Flow-through, (IP) immunoprecipitation. (B, left, upper and lower panels) RP in inflorescences using two different protocols without or with sucrose cushion respectively; see main text for details. (Right panel) RP on 15%–60% sucrose gradients was conducted in inflorescences. AGO1 and selected miRNA analyses were conducted as in A. (C) RP as described in B, but in WT or hen1-6 inflorescences, in the presence of 40 mM EDTA or RNase A/T1. (D) Western analysis of AGO1 upon RP conducted as in B in amp1-30i Arabidopsis. Immunodetection of ribosomal protein S14 was used as a control in B–D to verify RP quality. All these experiments were produced in triplicate.

FIGURE 2.

miRNA steady-state levels correlate with their association with polysomes. (A) Read size distribution in reads per million (RPM) grouped by genomic annotation for monosomal (Mono1 and Mono2), polysomal (Poly1 and Poly2), and TRAP sRNA-sequencing libraries. Correlations between mature miRNA accumulations in polysomal fraction versus inflorescences and TRAP versus inflorescences are shown in the lower right corner, with selected miRNAs indicated in red. (B) TRAP and RP were conducted in parallel as in Figure 1A,B to monitor the abundance of tasi-, mi- and siRNAs in ribosomal complexes via northern analysis using labeled complementary oligonucleotide or random-primed PCR-amplified fragments as probes, respectively. For RP, varying inputs of total RNA, as indicated, were loaded onto the sucrose gradients.

FIGURE 3.

In silico identification of pseudogene derived siRNAs. (A) Western analysis of AGO1, AGO2, AGO4, AGO5, and AGO10 upon cosedimentation on monosomes and polysomes via RP, as in Figure 1B. The ribosomal protein S14 was used as a control to verify the RP quality. This experiment was produced in triplicate. (B) Ratios of unique-mapper 24-nt siRNAs versus total 24-nt siRNAs, grouped by genomic annotation of sRNAs from publicly available sequencing libraries of total RNA and AGO IPs. (C) Expression heat-map of pseudogene derived siRNA parent genes at the various developmental stages encompassed in the bar.toronto database, using unsupervised hierarchical clustering. (D) Boxplot representation of average cytosine methylation levels in the three contexts for parent genes and respective pseudogenes of cluster 1 or cluster 2. (E) mRNA steady-state accumulation in polysomal and monosomal fractions based on RNA-seq conducted in the indicated libraries, for the parent genes and respective pseudogenes of cluster 1 or cluster 2. (F) Total 21-to-24 nt sRNA accumulation profiles for psARK1 and psPEROS pseudogenes, in polysomal and monosomal fractions. (G) Boxplots representation of average GC contents for parent genes and respective pseudogenes in cluster 1 or cluster 2.

FIGURE 4.

Tissue-specificities, biogenesis and loading of various pseudogene derived siRNA classes. (A) Northern analysis of specific pseudogene derived siRNA populations in the indicated genetic backgrounds from inflorescences; the hybridization probes were random-primed, PCR-amplified DNA fragments covering the most abundant pseudogene derived siRNA signal detected in sRNAseQ analyses. (B) Same as in A but in various Arabidopsis tissues. sRNAs derived from REP2 and SIMPLE HAT were used as representatives of conventional rasiRNAs. (C) AGO1, AGO4, and AGO5 IPs were carried out from inflorescences and 1–3 DAP siliques, and the presence of the proteins in IP fractions was assessed by western analysis using antibodies against native proteins (left panel). Pseudogene derived siRNA populations co-IPed with the corresponding AGOs were detected by northern analysis as in B (right panel). Hybridization to the U6 small RNA was used as a loading control in panels A and B. A and B were produced in triplicate, C in duplicate.

FIGURE 5.

Class I, but not class II, pseudogene derived siRNAs cosediment in siliques’ polysomes. RP conducted in inflorescences, 1DAP and 3 DAP siliques. Fractions 1 and 2 (light fractions), fractions 4 and 5 (monosomes), and fractions 7, 8, and 9 (polysomes) were pooled to optimize northern analysis of the indicated pseudogene derived siRNA populations as in Figure 4A. This experiment was produced in triplicate.

FIGURE 6.

A model for the evolution and modes of action of Cluster 1 pseudogene derived siRNAs of class I.