Stem-Loop Recognition by DDX17 Facilitates miRNA Processing and Antiviral Defense

SummaryDEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements

Living or dying by RNA processing: caspase expression in NSCLC

Protein expression in humans is controlled by numerous RNA processing steps that occur between transcription of a gene and translation of protein. However, the importance of such regulatory steps to human diseases, especially cancer, is just now coming to light. Changes in the alternative splicing or stability of mRNA transcribed from genes involved in cell-cycle control, cell proliferation, and apoptosis has been linked to tumor formation and progression. Nevertheless, in the majority of these cases, the identity of the regulators that control the expression of such cancer-related genes is poorly understood. In this issue of the JCI, Goehe et al. demonstrate that heterogeneous nuclear ribonuclear protein family member L (hnRNP L), a member of the hnRNP family of RNA processing factors, is specifically phosphorylated in non–small cell lung cancer (NSCLC). The phosphorylated hnRNP L, in turn, promotes expression of the antiapoptotic form of caspase-9, thereby contributing to tumorigenesis

The τCstF-64 Polyadenylation Protein Controls Genome Expression in Testis

<div><p>The τCstF-64 polyadenylation protein (gene symbol <em>Cstf2t</em>) is a testis-expressed orthologue of CstF-64. Mice in which <em>Cstf2t</em> was knocked out had a phenotype that was only detected in meiotic and postmeiotic male germ cells, giving us the opportunity to examine CstF-64 function in an isolated developmental system. We performed massively parallel clonally amplified sequencing of cDNAs from testes of wild type and <em>Cstf2t^−/−</em> mice. These results revealed that loss of τCstF-64 resulted in large-scale changes in patterns of genome expression. We determined that there was a significant overrepresentation of RNAs from introns and intergenic regions in testes of <em>Cstf2t^−/−</em> mice, and a concomitant use of more distal polyadenylation sites. We observed this effect particularly in intronless small genes, many of which are expressed retroposons that likely co-evolved with τCstF-64. Finally, we observed overexpression of long interspersed nuclear element (LINE) sequences in <em>Cstf2t^−/−</em> testes. These results suggest that τCstF-64 plays a role in 3′ end determination and transcription termination for a large range of germ cell-expressed genes.</p> </div

Relative usage of distal poly(A) sites (RUD) decreases throughout testis development, but less so in <i>Cstf2t^−/−</i> mouse testes.

<p>The Y-axis is the mean RUD score which reflects relative usage of distal poly (A) sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048373#pone.0048373-Ji2" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048373#pone.0048373-Ji3" target="_blank">[27]</a>. RUD values were based on three replicates for the 17, 22 and 25 dpp time points and two replicates for the 85 dpp time point. Error bars indicate the standard deviation (P values for T-test are 0.69, 0.32, 0.006 and 0.15 for 17, 22, 25 and 85 dpp, respectively comparing KO and WT). A step-wise decrease can be seen, indicating progressive shortening of 3′ UTRs or more usage of proximal poly(A) sites from 17 to 85 days postpartum (dpp). However, <i>Cstf2t^−/−</i> differs from wild type starting at 25 dpp through 85 dpp.</p

ISGs are down-regulated and have increased read-through in <i>Cstf2t^−/−</i> mouse testes.

<p>(<b>A</b>) Cumulative frequency of microarray log₂ mRNA expression changes of <i>Cstf2^−/−</i> (KO25) versus wild type (WT25) mouse testis at 25 dpp. Short genes were defined as the lowest 20% in length, with a cutoff of 6658 bp or shorter. Indicated are long multi-exon genes (11,451 genes, blue), short multi-exon genes (2,324 genes, green), and short single-exon genes (541 genes, red). There are 276 short single-exon genes in the region between -2 and 0 log₂ expression change. P values are 4.2×10⁻⁴ between short single-exon and short multi-exon genes and 1.0×10⁻¹⁵ between long multi-exon and short-multi-exon genes by a K-S test. (<b>B</b>) qRT-PCR was performed using primers specific for the indicated genes (see Table S1) normalized to <i>Rps16</i>. Each bar represents the amount (in percent) of the indicated mRNA in 25 dpp <i>Cstf2t^−/−</i> mouse testis RNA compared to wild type. The asterisks indicate values that are significantly different (P<0.001) from Rsp16 and Actb by ANOVA (Bonferroni multiple comparisons test). (<b>C</b>) Polyadenylation read-through assay. Random-primed cDNA is made from RNA from wild type or <i>Cstf2t^−/−</i> mouse testes. qRT-PCR is then performed using primer pairs within the body of the gene (“Upstream”) or downstream of the polyadenylation site (“Downstream”). An increase in read-through is measured as in increase in the downstream value compared to the upstream value in <i>Cstf2t^−/−</i> mice after normalization. (<b>D</b>) Read-through increases for ISGs in <i>Cstf2t^−/−</i> mouse testes. The polyadenylation read-through assay described in (C) was performed on the indicated genes and normalized to 1.0 in the wild type mice. The asterisk (P<0.05) and double asterisk (P<0.01) indicate values that differ significantly from the wild type by a Student's t-test.</p

High-throughput cDNA sequencing (RNA-seq) finds significant differences between wild type and <i>Cstf2t^−/−</i> mouse testis RNAs.

<p>(<b>A</b>) RNA was pooled from testes of five 25 dpp mice of either wild type or <i>Cstf2t^−/−</i> genotype, cDNA synthesized, and high-throughput sequencing performed (see Materials and Methods). (<b>B</b>) RNA-seq from wild type (∼55,000 reads) and <i>Cstf2t^−/−</i> (∼77,000 reads) mouse testis samples were not biased when mapped to the mouse genome. 454 sequencing reads were mapped to the mouse genome (Mouse Genome Assembly version mm8) using BLAT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048373#pone.0048373-Kent1" target="_blank">[24]</a>. Pie graphs show that similar proportions of reads mapped to either unique genomic regions (blue), multiple regions (non-unique, green), or could not be mapped to known regions (unmapped, tan) in samples from wild type or <i>Cstf2t^−/−</i> mouse testes. The proportion of uniquely mapped reads has no statistical difference between wild type and Cstf2t−/− mice (85.4% vs. 85.2%; P = 0.14, Fisher's exact test). (<b>C</b>) Introns and intergenic regions were more highly expressed in testes of <i>Cstf2t^−/−</i> mice, while exons were less expressed. Pie graphs show percentages of reads that were uniquely mapped to different regions of the genome for wild type and <i>Cstf2t^−/−</i> mice. Exon (blue), reads fully aligned to exons; exon & intron (green), reads aligned to both exonic and intronic regions; intron (tan), reads fully aligned to introns; 3′ UTR-ext (orange) and 5′ UTR-ext (purple), reads aligned to within 4 kb downstream of 3′ UTR or 1 kb upstream of the 5′ UTR, respectively; intergenic (grey), reads aligned to regions not within annotated genes or their extended regions. The difference of proportion of reads mapped to different genomic regions is significant: P <10–323 for both the intergenic region and intronic region (Fisher's exact test, exon region used as control).</p

LINE-1 mRNAs are over-represented in <i>Cstf2t^−/−</i> mouse testes.

<p>(<b>A</b>) Comparison of reads uniquely mapped to different transposable elements in intergenic regions in 25 dpp wild type (blue bars) and <i>Cstf2t^−/−</i> (green bars) mice. Error bars denote standard deviations. LINE-1, SINE and LTR classes of transposable elements were identified using RepeatMasker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048373#pone.0048373-Lee1" target="_blank">[46]</a>. LINE-1 mRNAs are 2.1-fold more abundant in <i>Cstf2t^−/−</i> mice than in wild type (P = 9.0×10⁻¹⁴¹ by Fisher's exact test), while SINEs are only 0.8-fold different (P = 4.4×10⁻⁹) and LTRs are 1.05-fold different (P = 0.15). (<b>B</b>) Comparison of reads uniquely mapped to different transposable elements in intronic regions in wild type and <i>Cstf2t^−/−</i> mice. LINE-1 mRNAs are 1.7-fold more abundant in <i>Cstf2t^−/−</i> mice than in wild type (P = 5.3×10⁻²⁶), SINEs are 1.1-fold different (P = 1.6×10⁻³) and LTRs are 1.3-fold different (P = 4.8×10⁻⁵). (<b>C</b>) Comparison of reads mapped to different transposable elements in multiple genomic regions in wild type and <i>Cstf2t^−/−</i> mice. LINE-1 mRNAs are 5.1-fold more abundant in <i>Cstf2t^−/−</i> mice than in wild type (P = 1.1×10⁻¹⁷³), SINEs are 1.6-fold different (P = 1.2×10⁻⁵) and LTRs are 1.7-fold different (P = 1.3×10⁻¹¹). (<b>D</b>) Comparison of unmapped reads that align with transposable elements in wild type and <i>Cstf2t^−/−</i> mice. LINE-1 mRNAs are 5.2-fold more abundant in <i>Cstf2t^−/−</i> mice than in wild type (P = 1.1×10⁻¹¹²), SINEs are 1.0-fold different (P = 0.76) and LTRs are 2.2-fold different (P = 8.2×10⁻²¹). (<b>E</b>) Percent of uniquely mapped reads that partially or completely mapped to full length (≥6 kb) LINE-1 sequences in the genome in 25-dpp wild type (blue bars) or <i>Cstf2t^−/−</i> (green bars) mouse testes. LINE-1 mRNAs are 4.6-fold more abundant in <i>Cstf2t^−/−</i> mice than in wild type (P = 7.8×10⁻²², Fisher's exact text). Error bars denote standard deviations of proportion using the formula √[p*(1-p)/N] where p is the proportion of reads mapped to one type of repeat sequence and N is the total number of reads used for mapping. (<b>F</b>) Location of the 3′ end of the sequence reads in along uniquely-mapped full length LINE-1 sequences (from b). Each LINE-1 sequence was evenly divided into 5 regions from 5′ to 3′, and the number of reads whose 3′ ends mapped to each region was determined. The distribution of 3′ ends of reads of WT was significantly different than that of KO (P = 0.002, Chi-squared test).</p