Global Analysis of RNA Secondary Structure in Two Metazoans

The secondary structure of RNA is necessary for its maturation, regulation, processing, and function. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach to identify the paired (double-stranded RNA [dsRNA]) and unpaired (single-stranded RNA [ssRNA]) components of the Drosophila melanogaster and Caenorhabditis elegans transcriptomes, which allows us to identify conserved features of RNA secondary structure in metazoans. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals

Ars2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation

SummaryHere we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation

The role of RNA silencing in Drosophila antiviral immunity

Innate immune mechanisms are essential components of virus-host interactions and play a critical role in determining the outcome of infection. Cell-intrinsic antiviral pathways have been identified in humans, but the mechanisms governing their function are not fully understood. Moreover, arboviruses, which are transmitted by insect vectors, represent an emerging worldwide threat to human health, but insect antiviral pathways are only beginning to be uncovered. It is likely that additional factors involved in mediating conserved or insect-specific antiviral responses remain to be characterized. Therefore, I sought to identify novel host factors involved in antiviral immunity using image-based high-throughput RNAi screening in Drosophila cells. I found that a previously uncharacterized host factor Ars2 plays an essential role in restricting RNA viruses. Depletion of Ars2 from cells and flies renders them hypersusceptible to viral infection. Specifically, Ars2 controls infection at the level of RNA replication. One known antiviral pathway in insects that responds to viral RNA is RNA interference (RNAi). Using a variety of molecular and biochemical approaches, I found that Ars2 is important for RNAi-mediated silencing at the level of siRNA biogenesis. Additionally, I found that miRNA biogenesis is impaired in the absence of Ars2 in both Drosophila and mammals. Thus, Ars2 is a conserved component of RNA silencing that is critical for RNAi-mediated virus restriction in Drosophila.^ The Drosophila antiviral RNAi response is initiated by Dicer-2, which generates virus-derived siRNAs (vsiRNAs) from viral RNA. Viral dsRNA is thought to be the target of Dicer-2, although the precise viral precursors of vsiRNAs have not been well characterized. To determine the identity of these precursors, I employed small RNA deep sequencing of Drosophila cells infected with a diverse panel of viruses, and mapped the cloned vsiRNAs onto the corresponding viral genomes. Each virus generated a signature pattern of vsiRNAs, and these patterns suggest that dsRNA replication intermediates, structured single-stranded RNAs, and unique hairpins are all Dicer-2 targets. Therefore, the RNAi pathway is capable of recognizing a variety of viral substrates to mount a successful antiviral response.

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

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

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

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