Abstract
The ability to overcome cellular restrictions that exist for the export and translation of mRNAs with retained introns is a requirement for the replication of retroviruses and also for the expression of many mRNA isoforms transcribed from cellular genes. In some cases, RNA structures have been identified in the mRNA that directly interact with cellular factors to promote the export and expression of isoforms with retained introns. In other cases, a viral protein is also required to act as an adapter. In this report we describe a novel vector system that allows measurement of the ability of cis- and trans-acting factors to promote the export and translation of mRNAs with retained introns. One reporter vector used in this system is derived from an HIV proviral clone engineered to express two different fluorescent proteins from spliced and unspliced transcripts. The ratio of fluorescent signals is a measurement of the efficiency of export and translation. A second vector utilizes a third fluorescent protein to measure the expression of viral export proteins that interact with some of the export elements. Both vectors can be packaged into viral particles and be used to transduce cells, allowing expression at physiological levels from the integrated vector.
Highlights
Intron retention is an important form of alternative splicing that is prevalent during retroviral replication, and is found in the regulation of cellular genes
We describe a novel manipulated system that allows the detection and characterization of RNA elements and trans-acting protein factors that promote the nucleocytoplasmic export and translation of mRNAs with retained introns
In the case of complex retroviruses, such as HIV and equine infectious anemia virus (EIAV), there is considerable evidence that different viral isolates vary significantly in their www.nature.com/scientificreports ability to express intron-containing viral RNA and that these differences are important in pathogenesis and possibly in the regulation of viral latency[16,22,23,26,29]
Summary
Intron retention is an important form of alternative splicing that is prevalent during retroviral replication, and is found in the regulation of cellular genes. Special mechanisms have been described that promote the export and translation of mRNAs with retained introns[1] Simple retroviruses, such as the Mason-Pfizer monkey virus (MPMV), rely on an RNA element present in the transcript that retains an intron, which binds to a cellular protein complex consisting of Nxf[1] and the co-factor Nxt[1] to promote export and translation[2,3,4]. This element is referred to as a constitutive transport element (CTE) due to the lack of requirement for viral factors. Most existing assay systems have measured Rev-RRE function using transient transfection of non-lymphoid cell lines
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
