Unravelling the Tripartite Interactions Among Hepatitis E Virus RNA,miR-140 and hnRNP K |
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| Institution: | 1. Agharkar Research Institute, Nanobioscience Group, G. G. Agarkar Road, Pune 411004, India;2. Savitribai Phule Pune University, Ganeshkhind, Pune 411 007, India;1. Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213, USA;2. NIST Center for Neutron Research, Gaithersburg, MD 20899, USA;3. Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA;4. Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA;1. Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA;2. Department of Physics, University of Illinois at Chicago, Chicago, IL 60607, USA;1. LabISEN, Yncréa Ouest, 29200 Brest, France;2. University of Brest, Inserm, EFS, UMR 1078, GGB, 29200 Brest, France;3. CHRU Brest, Service de génétique, Laboratoire de génétique chromosomique, 29200 Brest, France;4. Centre de ressources biologiques, Site cytogénétique, CHRU Brest, 29200 Brest, France;1. Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Israel;2. Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida, USA;3. Paracelsus Medical University, Salzburg, Austria |
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| Abstract: | In the present investigation, we have identified the functional significance of the highly conserved miR-140 binding site on the Hepatitis E Virus (HEV) genome. Multiple sequence alignment of the viral genome sequences along with RNA folding prediction indicated that the putative miR-140 binding site has significant conservation for sequence and secondary RNA structure among HEV genotypes. Site-directed mutagenesis and reporter assays indicated that an intact sequence of the miR-140 binding site is essential for HEV translation. Provision of mutant miR-140 oligos carrying same mutation as on mutant HEV successfully rescued mutant HEV replication. In vitro cell-based assays with modified oligos proved that host factor-miR-140 is a critical requirement for HEV replication. Biotinylated RNA pulldown and RNA immunoprecipitation assays proved that the predicted secondary RNA structure of the miR-140 binding site allows the recruitment of hnRNP K, which is a key protein of the HEV replication complex. We predicted the model from the obtained results that the miR-140 binding site can serve as a platform for recruitment of hnRNP K and other proteins of HEV replication complex only in the presence of miR-140. |
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| Keywords: | Hepatitis E virus microRNA hnRNP K miR-140 |
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