Identification of novel microRNA-like molecules generated from herpesvirus and host tRNA transcripts

Abstract

We applied deep sequencing technology to small RNA fractions from cells lytically infected with murine gammaherpesvirus 68 (gammaHV68) in order to define in detail small RNAs generated from a cluster of tRNA-related polycistronic structures located at the left end of the viral genome. We detected 10 new candidate microRNAs (miRNAs), six of which were confirmed by Northern blot analysis, leaving four as provisional. In addition, we determined that previously identified and annotated viral miRNA molecules were not necessarily represented as the most abundant sequence originating from a transcript. Based on these new small RNAs and previously reported gammaHV68 miRNAs, we were able to further describe and annotate the distinctive gammaHV68 tRNA-miRNA structures. We used this deep sequencing data and computational analysis to identify similar structures in the mouse genome and validated that these host structures also give rise to small RNAs. This reveals a possible convergent usage of tRNA/polymerase III (pol III) transcripts to generate small RNAs from both mammalian and viral genomes.

FIG. 1.

Identification of novel candidate viral miRNAs in γHV68-infected fibroblasts. (A) Size-selected small RNA (18 to 30 nt) from 3T12 mouse fibroblasts infected with γHV68 was Solexa sequenced. The resulting reads were mapped to the γHV68 genome, and the first 7.5 kb of the genome is shown. The top line represents the position in the genome, and the annotated positive- and negative-strand ORFs are shown as green arrows. Yellow arrows represent annotated tRNAs on the positive strand, and blue arrows represent annotated miRNAs on the positive strand. Below that, the numbers of positive and negative strand reads per nucleotide are plotted on a log₂ scale. Thus, a peak represents all the reads in the region that overlap a particular nucleotide. Red arrows point to the position at which novel candidate miRNAs map. Data from three independent experiments were combined for the final analysis.

FIG. 2.

Predicted structure of novel candidate γHV68 miRNAs. Predicted secondary structures of tRNA-miRNA transcripts, with annotated miRNAs and peak reads depicted in red. Novel candidate miRNAs are boxed. Novel and previously annotated miRNAs were analyzed for peak read. #, annotated miRNAs for which the most abundant read is shifted slightly off and does not correspond to annotation. #*, an annotation that differs significantly from the peak read such that the peak read is on the opposite arm from miRNA annotation. Graphs depict read frequency across the nucleotide position of the structures for each polycistron. For tRNA7 and tRNA8, boxed regions depict areas with possible alternate folded structures.

FIG. 3.

Detection of candidate viral miRNAs. Northern blot analysis of novel candidate miRNAs from 3T12 cells mock infected (M) or infected with γHV68 (V). Ethidium bromide staining is shown to indicate RNA loading. Blots are representative of 2 or 3 independent experiments. In many cases, blots were probed for one miRNA and later stripped and reprobed for a different miRNA.

FIG. 4.

Detection of candidate viral miRNAs in vitro and in vivo. (A) RT-PCR detection of candidate miRNAs from lytically infected 3T12 cells. (B) RT-PCR detection of candidate miRNAs from bone marrow-derived macrophages infected with γHV68 for 20 h. (C) RT-PCR detection of candidate miRNAs from peritoneal cells isolated from Stat1KO mice infected with γHV68. Data represent results from two or three independent experiments. Error bars are standard errors of the mean. (D) Quantitative PCR products from mock or infected 3T12 cells described for panel A were run on a 3% NuSieve GTG agarose gel. Sizes of markers are indicated on the left. miRNA-amplified products run with the 80- to 89-nt markers.

FIG. 5.

Identification of novel tRNA-small RNA polycistrons in mouse. Sequencing data from Table 2 were also analyzed for mouse small RNAs associated with tRNAs. Three novel small RNAs (arrow) immediately downstream of tRNA717 on chromosome 1 (A), tRNA84 on chromosome 13 (B), and tRNA209 on chromosome 14 (C) were identified.

FIG. 6.

Detection of tRNA-small RNA polycistrons in mouse. (A) Predicted secondary structures of three mouse tRNA-small RNA transcripts. Nucleotides of the small RNAs are depicted in red. Blue lines indicate the sequences that were used to probe the Northern blots shown in panel B. (B) Northern blotting for tRNA-associated small RNAs was performed on RNA from mock-infected 3T12 (M), γHV68-infected 3T12 (V), and uninfected Vero (N) cells. Arrows and arrowheads indicate small RNAs, putative precursor structures, and possible polycistrons. Blots are representative of results from two independent experiments.