Plant Extracellular Vesicles Contain Diverse Small RNA Species and Are Enriched in 10- to 17-Nucleotide "Tiny" RNAs

Abstract

Small RNAs (sRNAs) that are 21 to 24 nucleotides (nt) in length are found in most eukaryotic organisms and regulate numerous biological functions, including transposon silencing, development, reproduction, and stress responses, typically via control of the stability and/or translation of target mRNAs. Major classes of sRNAs in plants include microRNAs (miRNAs) and small interfering RNAs (siRNAs); sRNAs are known to travel as a silencing signal from cell to cell, root to shoot, and even between host and pathogen. In mammals, sRNAs are transported inside extracellular vesicles (EVs), which are mobile membrane-bound compartments that participate in intercellular communication. In addition to sRNAs, EVs carry proteins, lipids, metabolites, and potentially other types of nucleic acids. Here we report that Arabidopsis (Arabidopsis thaliana) EVs also contain diverse species of sRNA. We found that specific miRNAs and siRNAs are preferentially loaded into plant EVs. We also report a previously overlooked class of "tiny RNAs" (10 to 17 nt) that are highly enriched in EVs. This RNA category of unknown function has a broad and very diverse genome origin and might correspond to degradation products.

Figure 1.

EVs are Enriched in TyRNA Sequences Derived from Diverse Sources. (A) Size distribution of sRNAs mapping to the genome; the percentage of each size class was calculated for each source of data, represented by the average of three replicate libraries. The x axis indicates the sRNA size and the y axis indicates its proportion. Left: sRNA sizes and proportions, as measured by total abundance of the reads; right: sRNA sizes and proportions, as calculated by distinct reads (unique sequences). (B) The abundance of reads mapping to nine different features of the Arabidopsis genome. These include the following from left to right: miRNA precursors; TAS precursors; snRNA and snoRNAs; rRNAs; tRNAs; TEs; CDSs (including introns and UTRs); intergenic sequences; and siRNA precursors dependent on Pol4. RPM, reads per million.

Figure 2.

EV tyRNAs Are Derived from Specific Regions of Precursor RNAs. (A) miRNA precursor structure, including from left to right, 5′ region, mature miRNA miR-5p, loop region, mature miRNA miR-3p, and 3′ region. (B) EV tyRNAs are enriched in sequences that map to the loop region of miRNA precursors. The abundance of sRNAs and tyRNAs mapping to different regions of a miRNA precursor, as described in (A). The y axis represents abundance, in reads per million (RPM). (C) EV tyRNAs mapping to mature miRNAs are cut out of the central region. Plots show the relative position of 5′ ends (left plot) or 3′ ends (right plot) of tyRNAs relative to nucleotide position within the parent miRNA. RPM, reads per million. (D) Mutations that affect abundance of specific subclasses of sRNAs have a corresponding influence on tyRNA abundance. The abundance of total leaf tyRNAs mapping to different features of the Arabidopsis genome. These include the following from left to right: miRNA precursors, TAS precursors, snRNA and snoRNAs; rRNAs; tRNAs; TEs; CDSs (including introns and UTRs); intergenic sequences; and known Pol4 precursors. The y axis represents abundance, in reads per million (RPM), in a logarithmic (Log₁₀) scale. Colored boxes highlight tyRNAs affected by mutations.

Figure 3.

tyRNAs that Map to a Single Position in the Genome are Enriched in Exon-Derived Sense-Strand Sequences. (A) The abundance of 1-hit tyRNAs mapping to different features of the Arabidopsis genome. RPM, reads per million. (B) The abundance of 1-hit tyRNAs mapping to 5′ UTRs, exons, introns, 3′ UTRs, and intergenic regions. The y axis represents abundance, in RPM. (C) The abundance of 1-hit tyRNAs mapping to different relative positions in mRNAs. The x axis represents the relative position in full-length mRNA, expressed in percentage of the total length, and the y axis represents abundance, in RPM. Note that scales are different for each plot.

Figure 4.

A Subset of sRNAs Display Differential Abundance in EVs Versus Total Leaf RNA or Apoplastic RNA. (A) Select miRNAs are enriched or depleted in EVs. Relative abundance of miRNAs in EVs and apoplast compared with abundance in total leaf RNA; the relative abundance is expressed as a heat map (see Key in center), with the samples being compared indicated below each heat map (*Q value ≤ 0.05 and **Q value ≤ 0.01). (B) Select siRNAs are enriched in EVs. Heat map indicates relative abundance of siRNAs (including phasiRNAs, tasiRNAs, and hc-siRNAs) in the indicated samples. Boxed tasiRNAs indicate sRNAs taken up by B. cinerea (shown in Cai et al., 2018; *Q value ≤ 0.05, **Q value ≤ 0.01, and ***Q value ≤ 0.001). (C) miRNA abundance does not correlate with EV enrichment. Absolute abundance in EVs of select miRNAs expressed in reads per million (RPM) on a logarithmic scale, including miRNAs enriched in EVs (orange) and those depleted in EVs (purple) relative to apoplastic RNA.