Shaima Akhlaq1, Neena G Panicker2, Pretty S Philip3, Lizna M Ali4, Jaquelin P Dudley5, Tahir A Rizvi6, Farah Mustafa7. 1. Department of Biochemistry, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address: 201570084@uaeu.ac.ae. 2. Department of Biochemistry, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address: ngpanicker@uaeu.ac.ae. 3. Department of Microbiology & Immunology, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address: prettys@uaeu.ac.ae. 4. Department of Microbiology & Immunology, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address: lizna@uaeu.ac.ae. 5. LaMontagne Center for Infectious Diseases, The University of Texas at Austin, 100 East 24th Street, NHB 2.616, Austin, TX 78712, USA. Electronic address: jdudley@austin.utexas.edu. 6. Department of Microbiology & Immunology, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address: tarizvi@uaeu.ac.ae. 7. Department of Biochemistry, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address: fmustafa@uaeu.ac.ae.
Abstract
BACKGROUND: The mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3' cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5' end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3' RmRE could facilitate translation of all other mRNAs, including gRNA. RESULTS: To address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner. CONCLUSIONS: MMTV contains a novel cis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.
BACKGROUND: The mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3' cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5' end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3' RmRE could facilitate translation of all other mRNAs, including gRNA. RESULTS: To address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner. CONCLUSIONS:MMTV contains a novel cis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.
Authors: Farah Mustafa; Dhuha Al Amri; Farah Al Ali; Noor Al Sari; Sarah Al Suwaidi; Preethi Jayanth; Pretty S Philips; Tahir A Rizvi Journal: PLoS One Date: 2012-10-16 Impact factor: 3.240