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Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes
Abstract:
Proteins that are located adjacent to the 5' end of mRNA in initiation complexes have been detected by chemical crosslinking. Reovirus mRNA containing radioactivity exclusively in the [3H]methyl-labeled "cap," m7G(5')ppp(5')-Gm, was oxidized with sodium periodate to convert the 2',3'-cis-diol of the 5'-terminal m7G to a reactive dialdehyde. Oxidized mRNA was incubated in cell-free protein-synthesizing systems derived from wheat germ or mammalian cells, and the resulting mRNA-ribosome initiation complexes were reduced with NaBH3CN. By this chemical procedure, putative Schiff bases between mRNA 5'termini and amino groups of neighboring proteins were stabilized by reduction, yielding covalently linked protein-RNA conjugates. Under conditions of ribosome binding, a limited number of polypeptides associated with the mRNA-ribosome complexes were crosslinked, suggesting that these proteins are positioned near and may interact with the 5' end of mRNA during initiation. This method should also be useful for studying the spatial relationships between molecules in other similar nucleoprotein complexes.
Insights
Researchers used chemical crosslinking to identify proteins near the 5' end of messenger RNA (mRNA) during initiation. This technique helps understand protein-RNA interactions in crucial cellular processes.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The 5' end of messenger RNA (mRNA) plays a critical role in initiating protein synthesis.
- Identifying proteins interacting with the mRNA 5' end is crucial for understanding translation regulation.
- Existing methods have limitations in precisely mapping protein proximity to mRNA termini.
Purpose of the Study:
- To develop and apply a chemical crosslinking method to identify proteins located near the 5' end of mRNA in initiation complexes.
- To investigate the spatial organization of proteins involved in mRNA-ribosome interactions during translation initiation.
Main Methods:
- Chemical modification of the 5'-terminal cap structure (m7GpppGm) of reovirus mRNA using sodium periodate to create a reactive dialdehyde.
- Incubation of oxidized mRNA in cell-free protein-synthesizing systems (wheat germ and mammalian) to form mRNA-ribosome initiation complexes.
- Reduction of Schiff bases formed between mRNA and protein amino groups using sodium cyanoborohydride (NaBH3CN) to stabilize covalent crosslinks.
Main Results:
- Successfully generated covalently linked protein-RNA conjugates, indicating successful crosslinking.
- Identified a limited number of polypeptides associated with mRNA-ribosome complexes that were crosslinked to the mRNA 5' end.
- These findings suggest specific proteins are positioned near the mRNA 5' terminus during initiation.
Conclusions:
- The developed chemical crosslinking method effectively identifies proteins proximal to the mRNA 5' end during translation initiation.
- This technique provides insights into the spatial arrangement of proteins within mRNA-ribosome complexes.
- The method holds potential for studying molecular interactions in other nucleoprotein complexes.