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. 2012 Aug;40(14):6787-99.
doi: 10.1093/nar/gks307. Epub 2012 Apr 9.

Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs

Zhihua Li  1 , Christine Ender, Gunter Meister, Patrick S Moore, Yuan Chang, Bino John
Affiliations

Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs

Zhihua Li et al. Nucleic Acids Res. 2012 Aug.

Abstract

Deep sequencing studies frequently identify small RNA fragments of abundant RNAs. These fragments are thought to represent degradation products of their precursors. Using sequencing, computational analysis, and sensitive northern blot assays, we show that constitutively expressed non-coding RNAs such as tRNAs, snoRNAs, rRNAs and snRNAs preferentially produce small 5' and 3' end fragments. Similar to that of microRNA processing, these terminal fragments are generated in an asymmetric manner that predominantly favors either the 5' or 3' end. Terminal-specific and asymmetric processing of these small RNAs occurs in both mouse and human cells. In addition to the known processing of some 3' terminal tRNA-derived fragments (tRFs) by the RNase III endonuclease Dicer, we show that several RNase family members can produce tRFs, including Angiogenin that cleaves the TψC loop to generate 3' tRFs. The 3' terminal tRFs but not the 5' tRFs are highly complementary to human endogenous retroviral sequences in the genome. Despite their independence from Dicer processing, these tRFs associate with Ago2 and are capable of down regulating target genes by transcript cleavage in vitro. We suggest that endogenous 3' tRFs have a role in regulating the unwarranted expression of endogenous viruses through the RNA interference pathway.

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Figures

Figure 1.
Figure 1.
Characteristics of small RNAs (sRNAs) matching to 5′ and 3′ termini of tRNAs. All positions are determined with respect to either the 5′ or 3′ ends of sRNAs/tRNAs. To determine the positions, the sRNAs (violet) and tRNAs (blue) are aligned by their 5′–3′ ends, as noted by respective illustrations. (A) The CCA motif preferentially occurs at the 3′ termini (position −1) of the sRNAs. To avoid artifacts from single sequences with high number of reads, only distinct sequences that contain CCA were used for the analysis. (B) The visualization of positions of all reads that map to tRNAs indicates that 3′ terminal of tRNAs produce the most number of fragments (3′ terminal peak). The two prominent peaks at ∼−60 and ∼−70 nt are due to 5′ specific processing of tRNAs that are ∼60 and ∼70 nt long, as illustrated in panel C. (C) Similar to tRNA 3′ specific reads, 5′ terminal of tRNAs are also specifically processed to yield RNA fragments (5′ terminal peak). The tRNA position (with respect to its 5′ end) at which the 5′ end of the sRNA reads are matched are used, as depicted by the bottom illustration. (D) Length distribution of 5′ (black) and 3′ (white) terminal RNAs from tRNAs indicate that 5′ terminal RNAs are predominantly 14−15 nt long, while 3′ terminal RNAs seem to manifest a broader range (∼16−18 nt).
Figure 2.
Figure 2.
Small terminal RNA fragments are produced preferentially from 5′ and 3′ termini of rRNAs, snoRNAs, snRNA, but not mRNAs. Reads (violet) that map to rRNA, snoRNAs, snRNAs and mRNAs are depicted as in Figure 1; positions are with respect to either the 5′ end (left panel) or the 3′ end (right panel) of the RNAs (blue) as illustrated. Subpanels with the characteristic 5′ or 3′ preferences are highlighted (asterisk) for clarity. For instance, snRNAs yield terminal RNAs exclusively from their 3′ end (right panel).
Figure 3.
Figure 3.
Terminal small RNA fragments from tRNAs, snoRNAs and rRNAs manifest a bias for either 5′ or 3′ processing. (A) Abundance of 5′ (solid blue) and 3′ (blue) terminal RNAs produced from individual tRNAs (X-axis). For clarity, illustration is limited to non-redundant tRNA sequences producing at least 10 reads (per million) of terminal RNAs. Similar patterns are observed for snoRNAs (B) and rRNAs (C). Profiles of snRNAs are omitted because they yield terminal RNAs exclusively from their 3′ end (Figure 2).
Figure 4.
Figure 4.
Processing of 5′–3′ terminal RNA fragments are conserved in mice. (A) Position of the small RNA reads relative to 5′ (left panel) and 3′ (right panel) termini of tRNAs. (B) Similar to human 5′–3′ terminal tRFs, the mouse tRFs preferentially yield either 5′ or 3′ fragments. (C) Asymmetric bias of abundant (≥10 reads/million) 5′ and 3′ tRFs is well preserved across different conditions [wild-type, Dicer/DGCR8 Knock outs (KO)] for each tRNA, with high Pearson correlation coefficients (0.89−0.97).
Figure 5.
Figure 5.
Presence of Ago2-associated 3′ tRFs that can cleave target RNA. (A) Total RNA from Dicer knockout mouse cells (Dicer−/−) or wild-type cells (Dicer+/+) were analyzed by northern blotting using probes specific for the 3′ of tRNAs (HisGTG and LeuCAG), and miR-125 b. (B) Endogenous Ago2 was immunoprecipitated from human HEK293 lysates, and the co-immunoprecipitated RNAs were extracted and analyzed by northern blots using probes against the 3′ tRFs (HisGTG and LeuCAG). GST antibody is used as control (C) Immunoprecipitated Flag-HA-Ago1 and Flag-HA-Ago2 were incubated with a 32P-cap-labeled target RNA (∼100 nt long), which contained a perfect complementary sequence to the 3′ termini of tRNAs (HisGTG and LeuCAG). Lanes indicated with T1 represent RNaseT1 digestions of the RNA substrates as ladders. The RNA sequence complementary to tRNA HIS-3′ and tRNA LEU-3′ is indicated by a black bar on the left.
Figure 6.
Figure 6.
Multiple RNases generate terminal tRFs from total RNA. (A) ANG (10 µg/ml) generates ∼20 nt long-terminal tRF as well as longer sitRNAs from total RNA extracts. In comparison an equal concentration of RNase A completely degrades the RNA within an hour, while a control lane with solution buffer (Buffer) generate terminal tRFs at much lower levels. (B) In vitro cleavage assay (t = 1 h) using total RNA from HEK293 cells (10 µg/lane) and different RNases at a series of concentrations indicate that at low concentrations, both RNase A and RNase I are able to produce similar terminal tRFs as that of ANG (arrows). In contrast, RNase T1 generates a different set of 3′ tRFs.
Figure 7.
Figure 7.
ANG processes tRFs within the TψC loop. (A) ANG can process in vitro transcribed, synthetic tRNA (untreated/buffer/ANG incubations at 0.5 h) to generate terminal tRFs. (B) Secondary structure of the in vitro-transcribed tRNA, highlighting the three mutations in TψC loop (M1−M3) and the tRF cleavage site (arrow). (C) ANG processing of the tRF is impaired by tRNA mutations in TψC loop (M1−M3).
Figure 8.
Figure 8.
3′ terminal tRFs might inhibit endogenous retroviral replication. (A) LTRs are highly enriched in complementary sequences of 3′ terminal tRFs. (B) The major tRNA terminal regions that produce abundant tRFs (reads ≥ 10, column 4) and their complementary LTR elements. (C) A potential model for the 3′ tRF pathway. The binding of 3′ tRF to the transcribed viral RNA could recruit double-stranded RNA specific endonucleases such as the highly efficient Ago2, enabling the rapid cleavage of the transcribed endogenous viral RNA.
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