VSV vsiRNAs are concentrated at the 5′ genomic terminus.

<p>(A) RNA species produced during VSV infection. (−) strand genome is depicted in blue, (+) strand antigenome and mRNAs in red. (B) VSV vsiRNA size distribution (control library). (C) Distribution of 21 nt VSV vsiRNAs across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) VSV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt VSV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) VSV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt VSV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

Replication of MHV in mice.

<p>Four-week old B6 mice were infected intracranially with 2000 pfu of MHV-A59 and sacrificed at the indicated days post infection. A) Brains were harvested and placed in gelatin saline, homogenized and infectious virus titered on mouse L2 fibroblasts. Titers shown are averages from five mice per group. B) Brains and C) spinal cords were harvested and RNA isolated on Qiagen RNeasy columns. qRT-PCR was performed to quantify relative abundance of MHV-A59 genomic and subgenomic RNA mRNA7. Data are plotted as means with SEM of 5-8 mice.</p

DCV genomic strand RNA is preferentially targeted by antiviral RNAi.

<p>(A) RNA species produced during DCV infection. (+) strand genome is depicted in blue, (−) strand antigenome in red. (B) DCV vsiRNA size distribution (control library). (C) Distribution of 21 nt DCV-derived vsiRNAs across the viral genome. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) DCV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt DCV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) DCV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt DCV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

A putative hairpin within the RVFV S segment generates abundant vsiRNAs in <i>Drosophila</i> and mosquito cells.

<p>(A) RNA secondary structure prediction of S segment IGR. The highly abundant vsiRNAs are mapped in red. (B) Northern blot analysis of RVFV-infected <i>Drosophila</i> DL1 cells, <i>Aedes aegypti</i> Aag2 cells, and <i>Aedes albopictus</i> C6/36 cells, probed for the S segment stem loop vsiRNAs and tRNA^val as a loading control.</p

Both the genomic and antigenomic RNA strands of the arbovirus RVFV generate vsiRNAs.

<p>(A) RNA species produced during RVFV infection. (−) strand genomic segments and mRNAs are depicted in blue, (+) strand antigenomes and mRNAs in red. (B) RVFV vsiRNA size distribution (control library). (C) Distribution of 21 nt RVFV vsiRNAs across the three viral genomic segments. vsiRNAs mapping to genomic strand are depicted in blue, antigenomic strand in red. (D) RVFV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt RVFV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) RVFV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt RVFV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

VACV terminal repeat-derived vsiRNAs are derived from long, repeat-containing precursors.

<p>(A) RNA secondary structure prediction of one of sixty 70-mer repeats located at the genomic termini. The abundant repeat-associated VACV vsiRNA is mapped in red. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s004" target="_blank">Figure S4</a>. (B) Expression analysis of VACV terminal repeat-associated transcripts in <i>Drosophila</i> DL1 cells and mouse embryonic fibroblasts (MEFs) by RT-PCR. The forward primer (red) lies within the 70-mer repeat sequence, while the reverse primer (green) binds a unique sequence outside of the repetitive region. The banding pattern of PCR products reflects the amplification of variable numbers of 70-mer repeats, as depicted in the diagram. M = DNA ladder.</p

RNA transcripts produced by VACV are targeted by the <i>Drosophila</i> RNA silencing pathway.

<p>(A) The VACV genome is a dsDNA molecule with covalently closed identical termini. (B) VACV vsiRNA size distribution (control library). (C) Distribution of 21 nt VACV vsiRNAs across the viral genome. vsiRNAs mapping to the (+) strand are depicted in blue, (−) strand in red. Black arrows mark genomic termini. (D) VACV vsiRNA size distribution between libraries depleted of RNase III enzymes. (E) Effect of RNase III enzyme depletion on 21 nt VACV vsiRNAs. vsiRNAs from control (black), Dcr-1 (orange), Dcr-2 (green) and Drosha (blue) depleted cells are compared. (F) VACV vsiRNA size distribution between libraries depleted of Argonaute proteins. (G) Effect of Argonaute depletion on 21 nt VACV vsiRNAs. vsiRNAs from control (black), Ago1 (orange), and Ago2 (green) depleted cells are compared. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055458#pone.0055458.s002" target="_blank">S2</a>.</p

Gene Ontology Analysis.

<p>The transcripts for which expression was significantly upregualted (894 transcripts) were analyzed using the functional annotation tool in DAVID and using only the molecular function, cellular component, and biological process terms in the gene ontology database. The most significant and non-redundant categories are represented here. The percentages of the 894 upregulated genes that are involved in each category are represented.</p

Nuclear m⁶A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development

<div><p>The <i>N</i>⁶-methyladenosine (m⁶A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m⁶A sites are found in the last exon and 3’ UTRs. Here we show that the nuclear m⁶A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; <i>Ythdc1</i>-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3’ UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m⁶A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3’ end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.</p></div

Expression and subcellular localization of YTHDC1 in oocytes and pre-implantation embryos.

<p>(A) YTHDC1 expression in adult mouse tissues. ACTB and TUBB (β-tubulin) served as loading controls. Heart and skeletal muscle contain little ACTB. (B) Western blot analysis of YTHDC1 in oocytes and pre-implantation embryos. TUBB served as a loading control. Note the lower levels of YTHDC1 in GV-stage oocytes, when normalized to TUBB. (C) Localization of YTHDC1 in oocytes and pre-implantation embryos. DNA was stained with Sytox green. Abbreviations: P5, P12: postnatal days 5, 12; GV, germinal vesicle stage; MII, metaphase II; 1C, 2C, 4C: 1-cell, 2-cell, 4-cell embryos; M/B, morula/blastocyst.</p