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Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
Published on: December 9, 2022
Mutational analysis of the predicted secondary RNA structure of the Mason-Pfizer monkey virus packaging signal
Farah Mustafa1, Kathy A Lew, Russell D Schmidt
1Department of Medical Microbiology, Faculty of Medicine and Health Sciences (FMHS), The United Arab Emirates University, P.O. Box 17666, Al Ain, United Arab Emirates.
Abstract:
The 5' end of the Mason-Pfizer monkey virus (MPMV) genomic RNA has been predicted to fold into a complex stem/loop structure that is thought to play a role in specific RNA encapsidation. In this study, we used a set of mutations that either abrogated or recreated the first four stem loops predicted within the 5' untranslated region (5' UTR) for effects on RNA packaging. Test of these mutations in our biological assay revealed that only stem loop 1 (SL1) was important for the packaging potential of MPMV, while mutations in none of the other stem loops affected packaging significantly. Interestingly, it was the primary sequence of SL1 RNA and not its secondary structure that affected packaging since compensatory mutations that reformed SL1 were unable to restore the packaging efficiency of the retroviral vector. Additionally, our mutational analysis reveals that stem loop 4, predicted to be the major packaging determinant of MPMV, does not seem to have a significant role in packaging. Finally, results of the biological effects of the structural mutations are discussed in relation to their effects on the folding potential of the various stem loops.
Insights
The Mason-Pfizer monkey virus (MPMV) RNA packaging relies on the primary sequence of stem loop 1 (SL1) within the 5' untranslated region (5' UTR), not its secondary structure. Stem loop 4 is not a major packaging determinant.
Area of Science:
- Virology
- Molecular Biology
- RNA Structure
Background:
- The 5' end of Mason-Pfizer monkey virus (MPMV) genomic RNA features predicted stem-loop structures.
- These structures are hypothesized to be crucial for specific RNA encapsidation.
Purpose of the Study:
- To investigate the role of the first four stem loops in the 5' untranslated region (5' UTR) of MPMV RNA in viral packaging.
- To determine whether RNA secondary structure or primary sequence is critical for packaging efficiency.
Main Methods:
- Site-directed mutagenesis was employed to disrupt and restore predicted stem loops (SL1-SL4) in the MPMV 5' UTR.
- A biological assay was used to assess the effect of these mutations on RNA packaging efficiency.
- Compensatory mutations were introduced to evaluate the role of secondary structure versus primary sequence.
Main Results:
- Only mutations affecting stem loop 1 (SL1) significantly impacted MPMV RNA packaging.
- Disrupting SL1 abolished packaging potential, while mutations in SL2, SL3, and SL4 had no significant effect.
- Restoring the SL1 secondary structure via compensatory mutations did not restore packaging efficiency, indicating the primary sequence is critical.
- Stem loop 4 (SL4), previously predicted as a major packaging determinant, showed no significant role in packaging.
Conclusions:
- The primary sequence of SL1 in the MPMV 5' UTR is essential for efficient RNA packaging.
- The secondary structure of SL1 is not the primary determinant for packaging.
- The role of SL4 as a major packaging determinant is not supported by these findings.
- These results refine our understanding of MPMV RNA packaging mechanisms.
