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RNA--protein interactions within the internal translation initiation region of encephalomyocarditis virus RNA
A V Borovjagin1, M V Ezrokhi, V M Rostapshov
1A.N. Belozersky Laboratory, Moscow State University, USSR.
Abstract:
Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.
Insights
Researchers identified two key protein binding sites on the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES). These proteins are crucial for IRES activity, influencing ribosome binding and protein synthesis initiation.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) is essential for cap-independent translation initiation.
- Understanding IRES-protein interactions is key to elucidating the mechanism of viral protein synthesis.
Purpose of the Study:
- To identify and characterize proteins that bind to the EMCV IRES.
- To investigate the functional significance of these protein-IRES interactions in translation initiation.
Main Methods:
- UV-cross-linking assays were used to detect protein binding to EMCV IRES derivatives.
- Mutagenesis and in vitro translation assays were employed to assess IRES activity.
- In vitro assembly of IRES transcripts was performed.
Main Results:
- Two proteins (p58 and p60) were found to bind to specific regions (nt 315-377 and nt 420-421) of the EMCV IRES.
- These binding sites form hairpin structures with a conserved UCUUU motif.
- Deletion of the p58 binding site significantly reduced IRES activity but did not abolish it.
- In vitro assembled IRES showed restored activity, highlighting the importance of specific binding sites.
Conclusions:
- The identified proteins (p58 and p60) play a significant role in EMCV IRES-mediated translation initiation.
- The UCUUU motif within hairpin structures is important for protein binding.
- The findings provide insights into the complex mechanism of cap-independent translation initiation by viral IRES elements.