Related Experiment Videos
Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5' noncoding region
J Pelletier1, G Kaplan, V R Racaniello
1Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Abstract:
Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.
Insights
Poliovirus mRNA translation is regulated by its 5' noncoding region. This region contains an inhibitory element that restricts translation in some systems but is overcome by a factor found in HeLa cell extracts.
Area of Science:
- Molecular Biology
- Virology
Background:
- Poliovirus mRNA has a long 5' noncoding region (NCR) with upstream AUG codons.
- Unlike most eukaryotic mRNAs, poliovirus mRNA uses a cap-independent translation mechanism.
Purpose of the Study:
- To investigate the role of the poliovirus 5' NCR in mRNA expression.
- To identify elements within the 5' NCR that regulate translation.
Main Methods:
- Construction and analysis of deletion mutants within the poliovirus 5' NCR.
- Assessment of translational efficiencies in various cell-free systems (reticulocyte lysate, wheat germ extract) and in Xenopus oocytes.
Main Results:
- The 5' NCR of poliovirus mRNA contains inhibitory cis-acting elements (nucleotides 70-381) that restrict translation.
- These inhibitory elements function specifically in reticulocyte lysate, wheat germ extract, and Xenopus oocytes.
- HeLa cell extracts contain trans-acting factors that overcome the inhibitory effects of the 5' NCR.
Conclusions:
- The poliovirus 5' NCR plays a critical role in regulating translation initiation.
- Differential expression of poliovirus mRNA is mediated by system-specific interactions between the 5' NCR and translation machinery.