tRNA-derived small RNAs target transposable element transcripts

Abstract

tRNA-derived RNA fragments (tRFs) are 18-26 nucleotide small RNAs that are not random degradation products, but are rather specifically cleaved from mature tRNA transcripts. Abundant in stressed or viral-infected cells, the function and potential targets of tRFs are not known. We identified that in the unstressed wild-type male gamete containing pollen of flowering plants, and analogous reproductive structure in non-flowering plant species, tRFs accumulate to high levels. In the reference plant Arabidopsis thaliana, tRFs are processed by Dicer-like 1 and incorporated into Argonaute1 (AGO1), akin to a microRNA. We utilized the fact that many plant small RNAs direct cleavage of their target transcripts to demonstrate that the tRF-AGO1 complex acts to specifically target and cleave endogenous transposable element (TE) mRNAs produced from transcriptionally active TEs. The data presented here demonstrate that tRFs are bona-fide regulatory microRNA-like small RNAs involved in the regulation of genome stability through the targeting of TE transcripts.

Figure 1.

Characterization of tRFs in Arabidopsis pollen. (A) Analysis of ncRNA-derived sRNAs in inflorescences and pollen. Values are averages of two independent bioreplicates. (B) Analysis of the relative accumulation of ncRNA-derived sRNAs in pollen relative to inflorescence (two bioreplicates for each tissue). Error bars represent standard deviation among biological replicates. The inflorescence mean value is normalized to 1.0. (C) tRF accumulation profile in different sRNA libraries derived from leaf (three bioreplicates), inflorescence (two bioreplicates) and pollen (five bioreplicates). (D) Northern blot detection of 18–19 nt AlaAGC 5΄-derived tRF and miR161 in pollen, inflorescence and leaf. (E) Cartoon representation of tRNA secondary structure with nucleotides that give rise to tRF-5s colored in orange. (F) Total RPM and fold-change heatmaps of 19 nt 5΄-derived tRF accumulation in pollen relative to inflorescence.

Figure 2.

Accumulation of 19 nt 5΄-derived tRFs is conserved in land plants. (A–C) tRF accumulation profile in pollen and leaf from rice (A) and maize (B), and from gametophore–sporophyte, protonemata and young gametophore–protonometa from Physcomitrella patens (C). (D and E) Total RPM (D) and fold-change (relative accumulation of 19 nt tRF-5s in reproductive compare to mostly somatic structures) heatmaps (E) of 5΄-derived 19 nt tRFs of Arabidopsis, rice and maize pollen and Physcomitrella patens gametophore–sporophyte.

Figure 3.

DDM1 influences the accumulation of tRFs. (A) tRF accumulation profile in ddm1 compared to wt Col inflorescence tissue. The average value of three bioreplicates is shown. (B) Distribution of 5΄ end nucleotide of 19 nt tRFs on tRNA loci in ddm1 inflorescence. (C) Northern blot detection of AlaAGC 19 nt tRF-5 in wt Col and ddm1 backgrounds. miR161 was used as loading control. (D) Total RPM and fold change heatmaps of 19 nt 5΄-derived tRF accumulation in ddm1 inflorescences and wt Col pollen both relative to wt Col inflorescence. (E) Northern blot detection of selected mature tRNAs (highlighted with an asterisk and in bold in panel D) in total RNA from wt Col and ddm1. U6 RNA was used as a RNA loading control. (F) Band intensity quantification in ddm1 relative to wt Col normalized to the U6 intensity for the Northern blots represented in E.

Figure 4.

5΄-derived tRFs have a microRNA-like biogenesis pathway. (A) tRF accumulation profile in ddm1 and ddm1/dcl1. (B) Northern blot detection of 19 nt AlaAGC tRF-5 in wt, dcl1 and ago1 pollen grains. (C) tRF enrichment profile in wt AGO1 immunoprecipitated sRNAs relative to accumulation in inflorescences. (D) Fold enrichment of tRFs and selected AGO1-loaded miRNAs or non-AGO1 loaded microRNAs (miR161.1a) (E) Northern blot detection of AlaAGC 5΄ 19 nt tRF in AGO1 immunoprecipitated sRNAs from wt Col, dcl1 and dcl2. (F) Northern blot detection of 19 nt AlaAGC tRF-5 in RNA extracted from cytoplasmic and nuclear fractions. miR163 and the mature form of tRNA Met were used as cytoplasmic RNA controls and the snRNA U6 was used as a control of nuclear RNA.

Figure 5.

tRFs target transposable elements. (A) Categorization of predicted tRF mRNA-targets. (B and C) wt Col inflorescence PARE read alignment along a 100 nt window 5΄ and 3΄ to the predicted target sites for protein coding genes (B) or TEs (C). (D) PARE read alignment along a 100 nt window 5΄ and 3΄ to the predicted target sites for TEs in wt and ddm1. (E) ddm1 tRF-targeted TEs super-family categorization and number of tRF target sites for tRF-targeted TEs in ddm1 (pie chart).

Figure 6.

tRF targeting validation. (A) tRF alignment with Athila6A. Lines indicate perfect complementarity, and dotted interactions specify imperfect non-canonical base pairing sites. Cleavage site and frequency of observed/number of 5΄RLM RACE PCR product sequences cloned are indicated over the black arrow. The red arrow indicates the PCR band of the expected size that was sequenced. 5΄ RLM RACE PCR cleavage site from Athila6A in the ddm1, ddm1/dcl1 and ddm1/ago1 background is below. (B) 5΄ RLM RACE PCR cleavage site from Athila6A in STTM transgenic lines sequestering the AlaAGC and MetCAT 19 nt tRF-5s. Sequestering the MetCAT tRF using the MetCAT-STTM transgene results in less Athila6A cleavage (top) and increased Athila6A steady-state mRNA levels assayed by qRT-PCR (bottom). Since the AlaAGC tRF does not target Athila6A, its sequestration by the AlaAGC-STTM transgene does not alter the cleavage of Athila6A (by the MetCAT tRF) (top), and therefore Athila6A does not accumulate steady-state mRNA (bottom). qRT-PCR of Athila6A (bottom) utilizes primers spanning the cleavage site, and the asterisk represents P < 0.05. (C) Cartoon representation of the transgene constructs used. KRP6 = pollen vegetative cell specific promoter, H2B-GFP = nuclear localized Green Fluorescent Protein. (D) Representative pictures of GFP intensity in pollen grains from wt Col or dcl1 plants carrying the transgene from panel C. (E) Box plot representation of relative GFP intensity values in wt Col or dcl1 pollen grains from transgenic lines expressing a construct with a ‘mock’ or MetCAT target site. Values over the boxes represent the percentage of pollen grains with detectable GFP (equivalent to segregation) in the T1 generation. Bars between samples indicate the P value as determined by Student's t-test (two tailed, 95% of confidence interval). N = number of distinct transgenic events assayed.