Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs

Abstract

Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, we showed that the processing of small RNAs derived from tRNA(Gln) is dependent on Dicer in vivo and that Dicer cleaves the tRNA in vitro.

FIGURE 1.

Length distribution of all sequence reads. (A) Plot shows the length distribution by abundance of each of the 22,836 unique sequences obtained from deep sequencing of HeLa cell extracts. (B) Length distribution of tRNA and miRNA matching reads. The length distribution of the sequence reads of classified by matching to either tRNA (dark gray bars) or miRNA (light gray bars).

FIGURE 2.

Box plot showing the distribution of specific processing scores, S_p. Only noncoding RNA types with an abundance per matching read of >20 (see Table 1) are plotted. The left and right of the boxes represent the first and third quartiles of the data (the interquartile range), and the bold line within the box is the median. The dotted lines represent 1.5 times the interquartile range and data points outside deemed to be outliers (open circles). The height of the boxes represent the amount of data there is with taller boxes having more data.

FIGURE 3.

5′ and 3′ processing variability of miRNA matching reads. The box plots detail the distributions of the percentage of reads that show extensions at the 5′ and 3′ ends for each of the miRBase annotated mature sequences. To avoid matching to degradation products, extensions at the 5′ end were limited to a maximum of two bases. Two types of extensions are determined for each end; when at least one of the extension bases match the hairpin miRNA sequence (Alt. end), and instances when one or more of the extended bases do not match the hairpin miRNA sequence (untemplated end).

FIGURE 4.

(A) Overview window from Jalview (Clamp et al. 2004; Waterhouse et al. 2009) of sequence reads aligning to a known tRNA sequence as determined by Vmatch. Sequence reads are color coded by abundance with red >500 reads and blue <10 reads. The majority of the reads align to the 5′ end of the tRNA. A small number of complementary sequences can be seen aligning to the 3′ end: tRNA*. (B) Sequence motif logo for tRNA 8mer sequence around the 19–20 position of selective cleavage. Nonredundant set of tRNAs with matching read abundances of at least 2000 (B), and a nonredundant set of all human tRNAs (C). Arrow indicates the tRNA cleavage site. Images were created with WebLogo (Crooks et al. 2004).

FIGURE 5.

tRNA abundance by amino acid type and anticodon usage. Scatterplots showing the relationship between tRNA read abundance and the number of unique transcripts for each amino acid type (A) and anticodon usage (B). The number of known transcripts is for all sequence unique transcripts.

FIGURE 6.

Small RNA derived from tRNA^Gln. (A) Putative folding of tRNA^Gln, arrow shows the predicted cleavage site. (B) Size distribution of small RNAs derived from tRNA^Gln. (C) Northern hybridization shows that a ∼20-nt-sized fragment derived from tRNA^Gln exclusively localized in the cytoplasm (right panel) while the let-7 miRNA also could be detected in the nucleoplasmic fraction (middle panel). The efficiency of cell fractionations was checked with Western blotting for cell fraction specific proteins (left panel; tubulin for cytoplasm, Lamin A/C for nucleoplasm).

FIGURE 7.

The processing of small RNAs derived from tRNA^Gln is Dicer dependent. (A) The processing of endogenous tRNA^Gln into small RNA could be stimulated with MgCl₂. S100 extract was supplemented with increasing concentration of MgCl₂ and incubated for 1 h at 37°C. The tRNA fragments were detected with Northern hybridization. (B–D) Dicer is required for the generation of the small RNA derived from tRNA^Gln. (B) Dicer was knocked down with siRNA in HeLa cells. The efficiency of the knock down was tested with Western blotting and tubulin was used as a loading control (left panel). Northern blots show the marked decrease of the tRNA-derived small fragment and the accumulation of pre-miRNA in the Dicer knocked down cells (right panel). U6 was used as a loading control. The relative abundance of pre-miR-21 and mature miR-21 to the U6 loading control was normalized to the RNA level of the control siRNA transfected cells. The result of the quantification is indicated on the top and the bottom of the Northern blot (right panel). (C) Similar experiment described in B, only Dicer was knocked down in 293 cells with doxycyline (Dox.) inducible shRNA. (D) Recombinant Dicer was incubated with uniformly labeled tRNA^Gln, and the products were separated in 15% denaturing gel. “–,” RNA incubated with reaction buffer only; “+,” independent processing experiments; “arrows,” tRNA-derived small RNAs; and “*,” a nonspecific fragment present in the mock Dicer processing assay.

FIGURE 8.

Small RNAs derived from tRNA^Gln poorly associate with Argonaute proteins. (A) small tRNA^Gln RNAs partially cofractionate with Ago2. HeLA S100 extract was fractionated through a Superdex-200 column. Every second fraction was tested for Ago2 and Dicer with Western blotting, for miR-21 and small tRNA^Gln (labeled as tRNA) species with Northern hybridizations. Fractions containing the corresponding size markers (kDa) are indicated with arrows. Vertical bars are representing the level of small tRNA in the corresponding fractions in arbitrary units. (B) RNAs derived from tRNA^Gln incorporate into Ago1 and Ago2 complexes. FLAG immunoprecipitations were carried out from cells that were transfected with empty FLAG, FLAG∷Ago1, and FLAG∷Ago2 fusion plasmids. The total lysate and the bound fractions were assayed with Northern hybridization using complementary RNA to the sequenced small tRNA^Gln fragment as a probe. Black arrows indicate the small RNAs derived from the tRNA. (C) small RNAs derived from tRNA^Gln are resistant to β-elimination. Northern hybridizations show the results of the effect of β-elimination on miR-21 and small RNA processed from tRNA. Black arrows indicate miRNA and small tRNA fragments.