TY - JOUR
T1 - RNA–RNA interactions between respiratory syncytial virus and miR-26 and miR-27 are associated with regulation of cell cycle and antiviral immunity
AU - Ressel, Sarah
AU - Kumar, Sujai
AU - Bermúdez-Barrientos, Jose Roberto
AU - Gordon, Katrina
AU - Lane, Julia
AU - Wu, Jin
AU - Abreu-Goodger, Cei
AU - Schwarze, Jürgen
AU - Buck, Amy H.
N1 - The authors thank Dr Alfonso Garrido-Lecca from the Yi group at the University of Boulder for sharing advice on modifications to the CLEAR-CLIP protocol. The authors thank the Macias group from the University of Edinburgh for sharing RT-qPCR primers, as well as the IgG control antibody. The authors also thank Dr Pablo Tristán-Ramos from the Heras group at the University of Granada for discussions around crosslinking conditions during the CLEAR-CLIP protocol optimisation and Dr Anton McCaffrey and Sabrina Shore from Trilink for discussions around the modified 3 adapter. Total RNA-seq library preparation and sequencing was carried out by the Genetics core at the Edinburgh Clinical Research Facility. Small RNA sequencing was carried out by Edinburgh Genomics at the University of Edinburgh. Author contributions: S.R. and A.H.B. designed the study. All authors participated in the discussion and interpretation of the results. S.R. and J.L. performed the experiments. K.G. optimised the CLEAR-CLIP protocol for the fluorescent 3 adapter. J.W. provided the virus and reagents. S.K. developed the CLEAR-CLIP bioinformatic pipeline in communication with S.R. and C.A.G. J.R.B.B. modified the CLEAR-CLIP pipeline. S.K., J.R.B.B., S.R. performed the bioinformatic analysis of the (small) RNA-seq data. J.R.B.B. and S.R. analysed the data with input from A.H.B. and C.A.G. S.R. and A.H.B. wrote the manuscript. All authors revised the manuscript.
PY - 2024/5/22
Y1 - 2024/5/22
N2 - microRNAs (miRNAs) regulate nearly all physiological processes but our understanding of exactly how they function remains incomplete, particularly in the context of viral infections. Here, we adapt a biochemical method (CLEAR-CLIP) and analysis pipeline to identify targets of miRNAs in lung cells infected with Respiratory syncytial virus (RSV). We show that RSV binds directly to miR-26 and miR-27 through seed pairing and demonstrate that these miRNAs target distinct gene networks associated with cell cycle and metabolism (miR-27) and antiviral immunity (miR-26). Many of the targets are de-repressed upon infection and we show that the miR-27 targets most sensitive to miRNA inhibition are those associated with cell cycle. Finally, we demonstrate that high confidence chimeras map to long noncoding RNAs (lncRNAs) and pseudogenes in transcriptional regulatory regions. We validate that a proportion of miR-27 and Argonaute 2 (AGO2) is nuclear and identify a long non-coding RNA (lncRNA) as a miR-27 target that is linked to transcriptional regulation of nearby genes. This work expands the target networks of miR-26 and miR-27 to include direct interactions with RSV and lncRNAs and implicate these miRNAs in regulation of key genes that impact the viral life cycle associated with cell cycle, metabolism, and antiviral immunity.
AB - microRNAs (miRNAs) regulate nearly all physiological processes but our understanding of exactly how they function remains incomplete, particularly in the context of viral infections. Here, we adapt a biochemical method (CLEAR-CLIP) and analysis pipeline to identify targets of miRNAs in lung cells infected with Respiratory syncytial virus (RSV). We show that RSV binds directly to miR-26 and miR-27 through seed pairing and demonstrate that these miRNAs target distinct gene networks associated with cell cycle and metabolism (miR-27) and antiviral immunity (miR-26). Many of the targets are de-repressed upon infection and we show that the miR-27 targets most sensitive to miRNA inhibition are those associated with cell cycle. Finally, we demonstrate that high confidence chimeras map to long noncoding RNAs (lncRNAs) and pseudogenes in transcriptional regulatory regions. We validate that a proportion of miR-27 and Argonaute 2 (AGO2) is nuclear and identify a long non-coding RNA (lncRNA) as a miR-27 target that is linked to transcriptional regulation of nearby genes. This work expands the target networks of miR-26 and miR-27 to include direct interactions with RSV and lncRNAs and implicate these miRNAs in regulation of key genes that impact the viral life cycle associated with cell cycle, metabolism, and antiviral immunity.
UR - https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE232686
UR - https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231788
UR - https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231787
UR - https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231784
U2 - 10.1093/nar/gkae116
DO - 10.1093/nar/gkae116
M3 - Article
SN - 0305-1048
VL - 52
SP - 4872
EP - 4888
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 9
ER -