Export of mRNAs to the cytoplasm most likely requires the interplay of many regulatory proteins. The HIV-1 encoded regulatory protein Rev may interact with some of these same factors as it chaperones unspliced and partially spliced transcripts to the cytoplasm. Rev mediates its activity through its interaction with a cis-acting element located on Rev-dependent transcripts, termed the RRE (Rev responsive element). Likewise, a cis-acting element, known as the constitutive transporter element (CTE) is present in simple retroviruses. The CTE alone can functionally substitute for Rev-RRE, to allow the transport of incompletely spliced transcripts. For Bridgette's thesis studies, we asked whether CTE and Rev/RRE elements utilize the same intracellular transport pathways. We propose a two pronged approach to answering this question. We propose to use a transient transfection assay, in which Gag expression is controlled either through the Rev/RRE system or through the presence of CTE on the transcript. If decreased levels of Gag expression are noted with increased concentrations of the competitors, then we would predict that the same intracellular pathways are utilized. In the second approach, we will repeat the above transfection conditions but monitor for the intracellular localization of the transcripts. We will monitor the localization of the CTE containing transcripts to see whether or not they co-localize with SC35. We will then determine if the addition of the competing transcript or proteins cause shifts in the distribution of these transcripts. These studies should answer the basic question of co-localization of these RNA pathways and provide a framework for Bridgette to continue her studies of RNA transport.
