LTR-Retrotransposon Control by tRNA-Derived Small RNAs

Abstract

Transposon reactivation is an inherent danger in cells that lose epigenetic silencing during developmental reprogramming. In the mouse, long terminal repeat (LTR)-retrotransposons, or endogenous retroviruses (ERV), account for most novel insertions and are expressed in the absence of histone H3 lysine 9 trimethylation in preimplantation stem cells. We found abundant 18 nt tRNA-derived small RNA (tRF) in these cells and ubiquitously expressed 22 nt tRFs that include the 3' terminal CCA of mature tRNAs and target the tRNA primer binding site (PBS) essential for ERV reverse transcription. We show that the two most active ERV families, IAP and MusD/ETn, are major targets and are strongly inhibited by tRFs in retrotransposition assays. 22 nt tRFs post-transcriptionally silence coding-competent ERVs, while 18 nt tRFs specifically interfere with reverse transcription and retrotransposon mobility. The PBS offers a unique target to specifically inhibit LTR-retrotransposons, and tRF-targeting is a potentially highly conserved mechanism of small RNA-mediated transposon control.

Keywords: LTR-retrotransposon mobility; epigenetic reprogramming; mouse endogenous retroviruses; small RNA; tRNA fragments; transposon control.

Figure 1. Stem cells with relaxed epigenetic control of LTR-retrotransposons express sRNA targeting ERVs, including 3′ tRNA-derived fragments (tRFs)

(A) Setdb1 knockout mouse ES cells and TS cells have elevated levels of LTR-retrotransposon (ERV) small RNA and (B) 3′ CCA tRFs which target ERVs. Boxplots represent reads per million total mapped reads (RPM) of biological replicates; for a list of all 33 sRNA libraries refer to Figure S3. Setdb1 knockout induced −/−, uninduced +/+; for details see STAR Methods section. (C) tRF-targeted ERVs (in TS cells) are potentially active and younger than the average genomic ERV copy (avg: average; mdn: median; ± SD). (D) The majority of ERV-K targeted by 3′ tRFs are from the ETn and IAP families which are the most active LTR-retrotransposons in mouse (average RPM values of 4 replicate Setdb1 −/− and 7 replicate TS sRNA libraries). For comparison, relative abundance of ERV-K sequences in the mm9 mouse genome: 9% belong to the IAP family, 1% to the ETn family. See also Figure S1.

Figure 2. tRFs targeting LTR-retrotransposons (ERV) in mouse are derived from the 3′ end of mature tRNAs

(A) Alignment of all expressed CCA-tRFs (one representative TS cell sample) along tRNA coordinates (position 0 = CCA end) reveals cleavage of mature tRNAs precisely 22 nt and 17–19 nt from the 3′ CCA end. For tRNA alignments of all samples as well as analysis of other tRNA-derived fragments see Figures S2 and S3. (B) tRNA cleavage sites and nomenclature according to Kumar et al. (2014) are indicated. (C) Size distribution of CCA-tRFs targeting ERVs shows the dominant fragment length in mouse stem cells is 18 nt.

Figure 3. 3′ tRFs match the primer binding site (PBS) of LTR-retrotransposons

(A) CCA tRF sequencing reads are complementary to the highly conserved PBS site of LTR-retrotransposons. Major targets are ETn and IAPEz (all genomic loci convoluted, one representative TS and Setdb1 −/− sample out of all replicates). (B) Life cycle of LTR-retrotransposons and -viruses. The long terminal repeats (LTR) encode promoter elements and termination signals. The RNA transcript contains a region repeated at either end (R), a 5′ unique segment (U5), and a segment only included at the 3′ end of the RNA (U3). The 3′ end of cellular tRNAs (red cloverleaf) primes reverse transcription by hybridizing to the primer binding site (PBS). After this segment has been copied into first-strand cDNA (brown line), the RNaseH activity of reverse transcriptase (RT) degrades the complementary RNA and the elongating cDNA is transferred to the 3′ end of the retrotransposon transcript hybridizing to the R region. The remaining RNA is partially degraded by RNaseH leaving behind primers for second-strand cDNA synthesis. After a second transfer event, first- and second-strand synthesis can be completed to result in a full-length, double-stranded retroviral DNA that will be integrated into the host genome.

Figure 4. 3′ CCA tRFs inhibit retrotransposition

(A) The plasmid based retrotransposition assay. Transcription of MusD and ETn is driven by a CMV promoter upstream of the R and U5 portion of the LTR needed for retrovirus replication (see Figure 3B). After transcription and splicing, the retroviral gene products reverse transcribe the retroviral RNA and integrate the cDNA into the host genome. The neomycin (neo) gene becomes active only after splicing and reverse transcription, so that each neo-resistant cell must have had a retrotransposition event. (B) To test for regulation by tRFs, 18 nt tRFs were cotransfected together with MusD and the non-autonomous, neo-marked ETn or together with an autonomous, neo-marked IAP reporter plasmid. IAP transcription is driven by its endogenous promoter. The assays were done in human HeLa cells, as previously described (Dewannieux et al., 2004; Ribet et al., 2004). Neo-resistant clones were fixed, stained, and counted to measure retrotransposon activity. (C) MusD/ETn retrotransposition is inhibited by tRFs against ETn while unaffected by an unrelated 3′ CCA tRF sequence or non-targeting RNA. (D) IAP retrotransposition is strongly inhibited by tRFs targeting IAP but not by an unrelated 3′ CCA tRF sequence. Colony counts are the mean of two replicates ± SD.

Figure 5. 18 nt 3′ tRFs do not interfere with primary transcript or protein levels but inhibit reverse transcription

(A) MusD and ETn RNA transcript levels are not affected in cells which showed decreased MusD/ETn activity after transfection of ETn tRFs. Mean transcript levels ± SD were determined by quantitative Taqman RT-PCR. (B) MusD Gag protein levels are not affected by co-transfection of 18 nt, targeting tRFs. (C) Uncapped 5′-P RNA ends were sequenced by high-throughput, modified RACE. Each track represents one replicate. The position of all 5′ RNA ends including RT-RNaseH products (boxed red) and potential RNA cleavage products within ~40 bp surrounding the PBS and 5′ UTR are shown. The decrease of RNaseH products indicates that 18 nt targeting tRFs specifically inhibit accumulation of retroviral RNA intermediates. Note that for visibility, y-axis RPM maxima differ between MusD, ETn, and tRF concentrations. Welch two sample t-test, p-value ** < 0.05, * < 0.1, ns = not significant (D) Downstream retroviral DNA intermediates are decreased by ETn tRFs. TaqMan primers and probe detect extrachromosomal, retroviral DNA only (position indicated in E). Data represented as mean ± SD, normalized to total transfected plasmid DNA. (E) Outline of ETn-neo retroviral intermediates and model of retrotransposon silencing by 18 nt tRFs. See also Figure S4.

Figure 6. MusD lacking a tRNA primer binding site is released from silencing by endogenous tRFs

(A) The MusD PBS was replaced by an unrelated sequence to destroy the tRF target site (MusD-PBS*). Relative increase of transposition was higher for low amounts of transfected transposon plasmids (here 25 ng) in agreement with endogenous level of tRFs being the effector. (B) MusD Gag protein and (C) MusD RNA level are higher in the MusD-PBS* mutant. (D) Likewise, luciferase reporter gene expression is released from silencing by endogenous tRFs when the PBS is mutated. The luciferase ORF was cloned exactly in place of the first MusD ORF. RLU = relative light units. (E) MusD protein expression is decreased by 22 nt ETn tRFs but not by control tRF oligos or 18 nt tRFs. (F) Retrotransposition efficiencies are affected according to silencing of coding competent MusD by 22 nt tRFs and inhibition of reverse transcription of ETn-neo by 18 nt tRFs. Colony counts are the mean of two replicates ± SD. p-values Welch two sample t-test, all data represented as mean ± SD. See also Figure S5 and S6.

Figure 7. 22 nt tRFs mediate post-transcriptional gene silencing while 18 nt tRFs interfere with reverse transcription including non-coding, mobile elements

Model of retrotransposon silencing by 3′ tRFs: 22 nt tRFs target coding-competent LTR-retrotransposons (here MusD) at the level of retroviral protein production. 18 nt tRFs inhibit reverse transcription of any element with perfect complementarity at the PBS, including non-autonomous elements (here ETn). tRFs specifically promote silencing of retrotransposition-competent elements which maintain a functional PBS.