Control of noncoding RNA production and histone levels by a 5' tRNA fragment

Abstract

Small RNAs derived from mature tRNAs, referred to as tRNA fragments or "tRFs," are an emerging class of regulatory RNAs with poorly understood functions. We recently identified a role for one specific tRF-5' tRF-Gly-GCC, or tRF-GG-as a repressor of genes associated with the endogenous retroelement MERVL, but the mechanistic basis for this regulation was unknown. Here, we show that tRF-GG plays a role in production of a wide variety of noncoding RNAs-snoRNAs, scaRNAs, and snRNAs-that are dependent on Cajal bodies for stability and activity. Among these noncoding RNAs, regulation of the U7 snRNA by tRF-GG modulates heterochromatin-mediated transcriptional repression of MERVL elements by supporting an adequate supply of histone proteins. Importantly, the effects of inhibiting tRF-GG on histone mRNA levels, on activity of a histone 3' UTR reporter, and ultimately on MERVL regulation could all be suppressed by manipulating U7 RNA levels. We additionally show that the related RNA-binding proteins hnRNPF and hnRNPH bind directly to tRF-GG, and are required for Cajal body biogenesis, positioning these proteins as strong candidates for effectors of tRF-GG function in vivo. Together, our data reveal a conserved mechanism for 5' tRNA fragment control of noncoding RNA biogenesis and, consequently, global chromatin organization.

Keywords: epigenetics; histones; tRNA fragment.

Figure 1.

tRF-Gly-GCC directs chromatin-mediated repression of MERVL-associated genes. (A) Metabolic labeling reveals transcriptional derepression upon tRF-GG inhibition. Genome browser tracks show total RNA levels, and newly synthesized RNAs obtained after 15 or 30 min of 4-thiouridine (4SU) labeling for ES cells transfected with esiRNAs targeting GFP, or with an LNA oligonucleotide antisense to tRF-GG. Effects of tRF inhibition on previously described MERVL-associated target genes (Macfarlan et al. 2012; Sharma et al. 2016) are nearly identical for total RNA as well as newly synthesized RNA (see also Supplemental Fig. S1B,C). (B) Increased accessibility at heterochromatin and weakly transcribed regions in tRF-GG-inhibited ES cells. Heat map shows log₂ fold change in ATAC-seq reads following tRF-GG inhibition, aggregated across the indicated types of chromatin (Bogu et al. 2016). (C) As in B, with tRF-GG effects on ATAC-seq occupancy and RNA abundance averaged across the indicated repeat elements. (D–F) Examples showing average ATAC-seq signal across the indicated genomic elements: RefSeq genes (D), MERVL elements (E), or MERVK elements (F).

Figure 2.

tRF-Gly-GCC represses expression of histone genes via the histone 3′ UTR. (A) mRNA abundance for two example histone genes—Hist1h1e (top) or Hist1h2bh (bottom)—in four replicate RNA-seq libraries from mock-transfected, GFP KD, and tRF-GG KD mES cells, as indicated. (B) Scatter plot comparing RNA abundance for histone genes (purple diamonds) and all other genes in GFP KD ES cells (X-axis) and tRF-GG-inhibited ES cells (Y-axis). Note that nearly all histone genes fall below the X = Y diagonal. (C) Cumulative distribution of the effects of tRF-GG inhibition on histone mRNA expression, with the Y-axis showing cumulative fraction of genes exhibiting any given log₂ fold change in expression (X-axis). Main panel shows data from murine ES cells (n = 4 replicates, KS P = 7.7 × 10⁻⁵), while inset shows data for human ESCs. See also Supplemental Figure S2. (D) qRT-PCR for Hist2h3b showing effects of transfecting the anti-tRF-GG LNA, or a synthetic tRF-GG oligonucleotide (bearing most of the modified nucleotides expected from human tRNA-Gly-GCC) (Materials and Methods). (E) tRF-GG inhibition leads to decreased histone protein levels. Western blots probed for Histone H4 or loading control Lamin B, as indicated. See also Supplemental Figure S2D,E. (F) tRF-GG regulates histone 3′ UTR-mediated reporter expression. We generated stable ES cell lines carrying a luciferase reporter bearing the 3′ UTR of Hist2h3b (Supplemental Fig. S4A shows data for an independent cell line bearing the Hist1h4j 3′ UTR). Bar graph shows average changes to reporter activity in response to control KD, tRF-GG LNA (14% decrease, P = 0.038), or the modified tRF-GG oligo (30% increase, P = 0.0002).

Figure 3.

tRF-Gly-GCC supports production of U7 and other noncoding RNAs. (A) Effects of tRF-GG inhibition on several gene families in human H9 ES cells. Individual dots show individual members of the indicated families, illustrating the widespread down-regulation of histone and snoRNA genes in response to tRF-GG inhibition. Ribosomal protein genes are shown as a representative highly expressed, but tRF-insensitive, gene family for comparison. Effects on several snoRNAs are independently validated by qRT-PCR and Northern blot in Supplemental Figure S5A,D,E. (B) Cumulative distribution plots showing tRF effects on the indicated gene families, as in Figure 2C. (C) Manipulating tRF-GG levels affects U7 snRNA production. ES cells were transfected either with LNA antisense oligos targeting tRF-Ser-GCT, tRF-Val-CAC, or tRF-GG, or with synthetic tRF-GG oligos either bearing appropriate modified nucleotides (modified) or lacking these modifications (unmodified). U7 levels were quantitated by Northern blot (n = 4) and normalized relative to 5S rRNA levels. Change in U7 levels is expressed relative to tRF-Ser-GCT inhibition (as an unrelated LNA control), revealing a significant (P = 0.03) decrease in U7 levels in response to tRF-GG inhibition, as well as modestly increased U7 levels in tRF-GG-supplemented cells. See also Supplemental Figure S5B,C,E. (D) Effects of tRF-GG KD on histone 3′ UTR reporters are suppressed by supplementation with additional U7 snRNA. ES cells were transfected with the LNA antisense to tRF-GG, with or without additional in vitro-synthesized U7 RNA. Effects of tRF-GG KD were significant (P = 0.0039 and 0.00013 for H3 and H4 reporters, respectively), while tRF-GG KD + U7 was statistically indistinguishable from control (P = 0.24 and 0.48, respectively). See also Supplemental Figure S6.

Figure 4.

tRF-Gly-GCC binds to hnRNPF/H. (A) Biotin-oligo pull-downs from murine ES cell extracts. Silver-stained gel shows two replicates each for pull-downs using biotin-tRF-GG or biotin-tRF-Lys-CTT, as indicated. Arrow indicates ∼50-kDa band enriched in tRF-GG pull-downs. (B) Domain architecture of hnRNPF and hnRNPH1. (C) Mass spec peptide counts for hnRNPH in control, tRF-Lys-CTT, or tRF-GG pull-downs. (D) Western blots show hnRNPF/H recovery following tRF-GG or tRF-Lys-CTT pull-down. Pull-downs were washed four times for 3 min with 50 mM Tris (pH 8.0) supplemented with 100 mM (W1), 250 mM (W2), or 500 mM (W3) NaCl. (E) Enrichment of tRF-GG and two control RNAs in hnRNPF/H immunoprecipitates (and IgG controls) from mESCs, expressed as percent of input. Data show average and standard deviation from six data points: two biological replicates with three technical replicates each. (F) Gel shift analysis of hnRNPH1 binding to tRF-GG. A synthetic oligonucleotide corresponding to tRF-Gly-GCC (GCAJULGUGGUUCAGUGGDAGAAUUCUCGC) was labeled at the 3′ end using fluorescein 5-thiosemicarbazide, then incubated at 3 nM in equilibration buffer (0.01% Igepal, 0.01 mg/mL carrier tRNA, 50 mM Tris at pH 8.0, 100 mM NaCl, 2 mM DTT) for 3 h along with increasing concentrations of purified hnRNPH1 protein from 1.35 nM to 2000 nM. See also Supplemental Figure S7. (G,H) Fit of gel shift binding data for tRF-Lys-CTT and tRF-GG. Fitting the binding data yields an estimated Kd of hnRNPH1 of ∼220 nM for tRF-GG, and >1 μM for tRF-Lys-CTT. (I) Fluorescence polarization data for hnRNPH1 incubations with labeled tRF-GG. Polarization values against the protein concentrations are fit to the Hill equation using Igor Pro software.

Figure 5.

hnRNPF/H represses the MERVL program. (A) Changes in the ES cell transcriptome following hnRNPF/H knockdown. Scatter plot shows mRNA abundance compared between control KD cells (X axis) and hnRNPF/H KD cells (Y axis) (Supplemental Table S4). (B) hnRNPF/H KD results in histone mRNA down-regulation. Cumulative distribution plot shows log₂ fold change (hnRNPF/H KD/Ctrl) for histone genes, and all other genes, as indicated. (C) hnRNPF/H suppresses ES cell entry into the MERVL-positive “2C-like state.” ES lines carrying a MERVL LTR-driven tdTomato (Macfarlan et al. 2012) were subject to control or hnRNPF/H KD (with or without synthetic tRF-GG), with bars showing mean ± standard deviation (n = 5 replicates) of the percentage of Tomato-positive cells. (D) hnRNPF/H is required for normal Cajal body morphology and gross chromatin architecture. Panels show typical images for the Cajal body marker coilin (green) and DAPI (blue), in control or hnRNPF/H KD ES cells. See also Supplemental Figure S8B. (E) Schematic of proposed mechanism for tRF-GG function. Our data suggest a model in which 5′ tRF-Gly-GCC supports production of a variety of noncoding RNAs in Cajal bodies, potentially downstream from binding to the hnRNPF/H proteins. Central to the current study is regulation of U7 snRNA production, which controls processing of the histone 3′ UTR via base pairing to the histone downstream element (HDE). Altered expression of histones then leads to downstream effects on the expression of MERVL-associated genes in murine embryonic stem cells and preimplantation embryos. The precise mechanism by which tRF-GG might support hnRNPF/H function remains to be elucidated; tRF-GG could stabilize hnRNPF/H leading to increased functional protein levels, or hnRNPF/H and tRF-GG could function together in a complex as depicted here.