TY - JOUR
T1 - A cis-Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability
AU - Akhlaq, Shaima
AU - Panicker, Neena G.
AU - Philip, Pretty S.
AU - Ali, Lizna M.
AU - Dudley, Jaquelin P.
AU - Rizvi, Tahir A.
AU - Mustafa, Farah
N1 - Funding Information:
This study was supported by United Arab Emirates University Zayed Center for Health Sciences and Terry Fox Funds for Cancer Research (Grants 31R020 and 21M095, respectively) and College of Medicine & Health Sciences (Grant NP 14-34) to T.A.R. and in part by United Arab Emirates University Zayed Center for Health Sciences (Grants 31R122 and 31R140, respectively) and College of Medicine & Health Sciences (Grant 31M331) to F.M. J.P.D. was funded by the National Institutes of Health (Grant R01 CA167053). Funding for open access charge: UAE University. The authors would like to thank Dr. Waqar Ahmad, Department of Biochemistry, College of Medicine & Health Sciences, United Arab Emirates University for his administrative assistance.
Funding Information:
This study was supported by United Arab Emirates University Zayed Center for Health Sciences and Terry Fox Funds for Cancer Research (Grants 31R020 and 21M095 , respectively) and College of Medicine & Health Sciences (Grant NP 14-34 ) to T.A.R. and in part by United Arab Emirates University Zayed Center for Health Sciences (Grants 31R122 and 31R140 , respectively) and College of Medicine & Health Sciences (Grant 31M331) to F.M. J.P.D. was funded by the National Institutes of Health (Grant R01 CA167053 ). Funding for open access charge: UAE University. The authors would like to thank Dr. Waqar Ahmad, Department of Biochemistry, College of Medicine & Health Sciences, United Arab Emirates University for his administrative assistance.
Publisher Copyright:
© 2018 The Authors
PY - 2018/10/19
Y1 - 2018/10/19
N2 - 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.
AB - 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.
KW - 5′ RmRE
KW - MMTV
KW - RNA stability element
KW - Rem
KW - transcript elongation
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U2 - 10.1016/j.jmb.2018.08.025
DO - 10.1016/j.jmb.2018.08.025
M3 - Article
C2 - 30179605
AN - SCOPUS:85054022249
SN - 0022-2836
VL - 430
SP - 4307
EP - 4324
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 21
ER -