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Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome
1Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305, USA.
Abstract:
Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.
Insights
Internal ribosome entry sites (IRES) function in natural cellular mRNAs, even when cap-dependent translation is blocked. This allows synthesis of essential proteins like eIF4G during viral infections.
Area of Science:
- Molecular Biology
- Virology
- Gene Expression
Background:
- Internal ribosome entry sites (IRES) are known in viral and some cellular mRNAs.
- Their function within natural, capped cellular mRNAs, especially during stress, is less understood.
Purpose of the Study:
- To investigate the functionality of IRES elements in their native capped cellular mRNAs.
- To determine if IRES-mediated translation persists when cap-dependent translation is inhibited.
Main Methods:
- Polysomal association and translation assays were performed on IRES-containing cellular mRNAs.
- Experiments were conducted in cells infected with poliovirus to inhibit cap-dependent translation.
- cDNA expression library screening was used to isolate and test eIF4GI mRNA sequences.
Main Results:
- Several known IRES-containing mRNAs (e.g., BiP, c-myc) remained associated with the translation apparatus and were translated during poliovirus infection.
- eIF4GI and eIF4GII mRNAs were also found in polysomes in infected cells.
- The 5'-end of eIF4GI mRNA demonstrated IRES activity in a dicistronic mRNA assay.
Conclusions:
- IRES elements are functional in their natural capped cellular mRNAs, even under conditions that block cap-dependent translation.
- eIF4G proteins can be synthesized via IRES-mediated translation during viral infections.
- This suggests a role for eIF4G in both cap-dependent and cap-independent translation initiation.