tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner

Abstract

tRNA related RNA fragments (tRFs), also known as tRNA-derived RNAs (tdRNAs), are abundant small RNAs reported to be associated with Argonaute proteins, yet their function is unclear. We show that endogenous 18 nucleotide tRFs derived from the 3' ends of tRNAs (tRF-3) post-transcriptionally repress genes in HEK293T cells in culture. tRF-3 levels increase upon parental tRNA overexpression. This represses target genes with a sequence complementary to the tRF-3 in the 3' UTR. The tRF-3-mediated repression is Dicer-independent, Argonaute-dependent, and the targets are recognized by sequence complementarity. Furthermore, tRF-3:target mRNA pairs in the RNA induced silencing complex associate with GW182 proteins, known to repress translation and promote the degradation of target mRNAs. RNA-seq demonstrates that endogenous target genes are specifically decreased upon tRF-3 induction. Therefore, Dicer-independent tRF-3s, generated upon tRNA overexpression, repress genes post-transcriptionally through an Argonaute-GW182 containing RISC via sequence matches with target mRNAs.

Keywords: post-transcriptional gene regulation; small noncoding RNA; tRF; tRNA fragments.

FIGURE 1.

tRF-3s have distinct lengths and interact with Argonaute. (A) tRNA secondary structure depicting the tRF-3 cleavage sites. (B) Read counts for tRF-3s in AGO PAR-CLIP data (Hafner et al. 2010). Individual tRF-3s are arrayed along the x-axis with their expression levels (number of reads per million mapped reads) shown on the y-axis. The tRF-3s studied in this paper are indicated. (C) Number of reads per million mapped reads for each tRF-3 in top 300 chimeric reads from Ago-CLASH data (Helwak et al. 2013). (D) Mapped position along the length of the parental tRNA (x-axis) of small RNAs derived from that tRNA and their abundance (y-axis: number of reads found in library GSM416733). Small RNAs from tRNA LeuAAG chr16.tRNA16 (upper), tRNA-CysGCA chr17.tRNA26 (middle), and tRNA LeuTAA chr6.tRNA83 (lower) are shown, and the tRFs studied in this paper indicated. X-axis is the position on tRNA gene. Blue arrowhead and (***) indicate the end of mature tRNA and anticodon, respectively.

FIGURE 2.

tRF-3s produced by tRNA overexpression are loaded into Argonaute. (A) Relative levels of indicated tRNAs upon tRNA overexpression. Mean and S.D. of three independent experiments. (*) P-value <0.05 (Wilcoxon–Mann–Whitney Test). (B) Relative levels of indicated tRFs upon overexpression of the tRNAs indicated in (A) in the same order from left to right. Mean and S.D. of at least three independent experiments. (*) P-value <0.05 (Wilcoxon–Mann–Whitney Test). (C) Northern blot showing the tRF-3s produced after overexpression of the indicated parental tRNA. Lower panel shows equal loading of lanes. (D) Northern blot showing the association with Argonaute of tRF-3009a produced from tRNA overexpression. The immunoblot showing successful Argonaute immunoprecipitation is in Supplemental Figure S2.

FIGURE 3.

tRF-3s down-regulate target expression through complementarity in 3′ UTR of luciferase reporter. (A) Luciferase reporter assays using Renilla luciferase with a perfect complementary sequence to tRF-3 at the 3′ UTR. Renilla luciferase levels were first normalized to Firefly luciferase levels from the same transfection and then normalized to no tRNA/tRF overexpression (empty vector) control. (*) P-value <0.05 (t-test). (B) Degree of repression correlates with tRNA overexpression amount. Amount of transfected tRNA plasmid was titrated to measure the change in the degree of repression. Luciferase reporter assays were analyzed as described in A. (C) Luciferase reporter assay showing the specific repression by each corresponding tRF-3. (*) P-value <0.05 (t-test).

FIGURE 4.

Seed sequence is required for target repression by tRF-3s. Luciferase reporter assays with mutant target site at the luciferase reporter upon tRF-3001 (A), tRF-3003 (B), and tRF-3009 (C) overexpression. Canonical seed region on each tRF-3 and complementary sequence on each target are highlighted in yellow. Mutated regions are underlined and colored red. P-values are calculated by t-test comparing luciferase reporter with indicated target sequence to empty vector control (*) P-value <0.05; (**) P-value <0.005 (t-test).

FIGURE 5.

Target repression by tRF-3s is independent of Dicer but dependent on Argonautes. (A) Luciferase reporter assays after tRNA overexpression to produce tRF-3009 ± knockdown of Argonaute proteins. (*) P-value <0.05 (t-test). (B) Northern blot showing tRF-3009 levels in tRNA overexpressing cells after Ago knockdown. Lower panel shows equal loading of lanes. (C) tRF-3 read counts in small RNA sequencing data from WT and two different Dicer knockout clones of HEK293T. Small RNA sequencing data from Bogerd et al. (2014) (D) Luciferase reporter assays in wild-type and Dicer knockout HEK293T cells; NoDice 4–25 (middle), and NoDice 2–20 (right) (Bogerd et al. 2014).

FIGURE 6.

tRF-3s associate with GW182/TNRC6 proteins. (A) Read counts for miRNAs or tRF-3s in TNRC6A, TNRC6B, and TNRC6C PAR-CLIP data from Hafner et al. (2010). Each microRNA or tRF is given an arbitrary identifying number along the x-axis. The expression level (expressed as reads per million mapped reads) of each microRNA or tRF is shown on the y-axis. (B) Normalized positional T to C mutation frequencies for miRNA or tRF-3 reads found in the TNRC6A, TNRC6B, and TNRC6C PAR-CLIP data. (C,D) Complementarity in the target RNA cross-link-centered RNAs (CCRs) present in the TNRC6A PAR-CLIP to the 1–7, 2–8, 3–9, and 4–10-mer sequences from the 5′ end of the 20 most abundant microRNAs or tRF-3s seen in B. The CCRs are centered on the site of the U/C mutation and the number of targets with complementarity indicated at the corresponding base in the sequence of the CCR. For example, the maximum number of matches is seen with the 1–7 mer of the tRF and begins with the base immediately downstream from the cross-link site in the target RNA.

FIGURE 7.

Endogenous targets are repressed upon tRF-3009 overexpresssion by tRNA-LeuTAA transfection. (A) Luciferase assay upon tRNA overexpression to produce tRF-3009. In parallel, Renilla luciferase mRNA levels were measured by qRT-PCR from the same cells on which luciferase assays were performed (normalized to Firefly mRNA levels) (*) P-value <0.005. (B) Cumulative distribution function (CDF) plots showing the repression of tRF-3009 targets upon overexpression of tRNA producing tRF-3009. Targets have been predicted using RNA22 algorithm (Miranda et al. 2006). (C) Seed sequence complementarity in selected tRF-3009 targets that are identified in RNA-seq upon tRF-3009 expression. The number after 3′ UTR indicates the start position of the mRNA sequence match with 1 being the base immediately downstream from the stop codon. (Red line) Perfect base-pairing in seed; (black line) perfect base-pairing outside seed; (dashed line) wobble base-pairing. FER1 gene has two predicted complementary sites on its 3′ UTR. (D) Relative mRNA levels of indicated genes upon tRNA-LeuTAA transfection, leading to tRF-3009 overexpression. (*) P-value <0.05; (**) P-value, 0.005 (t-test). (E) Dual luciferase reporter assay on reporters containing the 3′ UTR of indicated genes after tRNA-LeuTAA transfection, leading to tRF-3009 overexpression. Perfect complementary sequence to the tRF-3009 serves as a positive control and all results are normalized to the “no site” reporter without any match to the tRF-3009 (*) P-value <0.05; (**) P-value, 0.005 (t-test).