Isabel Nawroth1,2,3, Florian Mueller4,5, Eugénia Basyuk3, Nancy Beerens2, Ulrik L. Rahbek2, Xavier Darzacq4, Édouard Bertrand3, Jørgen Kjems1,2, Ute Schmidt3
11Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark
22Interdisciplinary Nanoscience Center (iNANO), Aarhus University, 8000 Aarhus, Denmark
33Institut de Génétique Moléculaire de Montpellier?CNRS UMR 5535, 34293 Montpellier cedex 5, France
44Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, 75230 Paris cedex 05, France
55Institut Pasteur, Imaging and Modeling Unit, CNRS URA 2582, 75015 Paris, France
Tóm tắt
The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest that cotranscriptional formation of a stable export complex serves as a means to ensure efficient export of unspliced viral RNAs.
