Journal: Nucleic acids research

Article Title: RNA-RNA interactions between respiratory syncytial virus and miR-26 and miR-27 are associated with regulation of cell cycle and antiviral immunity.

doi: 10.1093/nar/gkae116

Figure Lengend Snippet: Figure 5. Pseudogenes and lncRNAs are HC miRNA targets and nuclear localisation of AGO2-miR-27 suggests potential roles in transcriptional gene regulation. ( A ) Enrichment of HC target sites in regulatory regions compared to non-filtered target sites. Target sites were filtered to remove sites o v erlapping 3 ′ UTRs and CDS. Shown are the mean ± SD for three Mock and four RSV CLEAR-CLIP replicates combined. Red asterisks indicate significant enrichment; One Brown-Forsythe and Welch ANO V A with Dunnett’s T3 multiple comparisons test. Due to low number of target sites in some of the categories, Mock and RSV samples were analysed together for more statistical power, * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0 0 01. ‘Overlapping’ indicates regulatory regions overlapping other genome annotations (pseudogenes (PSG), lncRNAs, introns, and other). (B ) Genome bro wser vie w of the genomic location of top miR-27 regulatory target o v erlapping a PSG, sho wing the conserv ation using Ph yloP100, features of the genomic locus, total RNA-seq co v erage in Mock and RSV-infected samples ( n = 3), as well as miRNA target sites coverage in Mock and RSV-infected CLEAR-CLIP samples. ( C ) RT-qPCR results for Malat1, RPL30, ZFAND5, ZFAND5 PSG lncRNA and miR-27a (left to right) le v els in cytoplasmic (cyto) and nuclear (nuc) fractions in Mock and RSV-infected cells. Data is shown as mean ± SD for two biological replicates. ( D ) Western blot analysis of AGO2 protein le v els in subcellular fractions sho wing whole cell ly sate (WCL), cytoplasmic (cyto), and nuclear (nuc) fractions f or Mock and R SV-infected cells. α-Tubulin and Calreticulin were used as cytoplasmic markers and Histone H3 as nuclear marker. ( E ) Quantification of AGO2 protein levels in (D). Data is shown as mean ± SD for four biological replicates and normalised to WCL Mock. ( F ) Western blot analysis of cellular fractionation of naïve A549 showing cytoplasmic fraction as well as the soluble and insoluble nuclear fractions. α-Tubulin and Calreticulin were used as cytoplasmic markers and Histone H3 as nuclear marker associated with chromatin. ( G ) RT-qPCR results of PSG lncRNA expression after treatment with 5 nM GapmeR targeting PSG lncRNA (anti-PSG lncRNA) or control. Shown are three biological replicates with mean ± SD. Significance was tested with unpaired t wo-t ailed t -test (* P ≤0.05). ( H ) Western blot analysis of CEACAM1 after treatment with anti-PSG lncRNA GapmeR, siRNAs against ZF AND5 (siZF AND5) and CEACAM1 (siCEACAM1) and controls compared to β-Actin loading control. ( I ) Quantification of CEACAM1 protein le v els from (H) corrected for β-Actin and normalised to Untreated control. Shown are two biological replicates with mean ± SD. Significance was tested with One Brown-Forsythe and Welch ANO V A with Dunnett’s T3 multiple comparisons test.

Article Snippet: Primary antibodies used in this study: AGO2 (1:1500, clone C34C6, Cell signalling, 2897), α- Tubulin (1:2000, Cusabio, CSB-PA004344), β-Actin (1:1000, Cell signalling, 4967), Calreticulin (1:2000, Cell signalling, 2891), CEACAM1 (1:20000, Abcam, ab235598), Histone H3 (1:2000, Cell signalling, 4499), ZFAND5 (1:1000, Sigma, HPA018129).

Techniques: RNA Sequencing, Infection, Quantitative RT-PCR, Western Blot, Marker, Cell Fractionation, Expressing, Control