Some picornaviral and cellular mRNAs initiate translation in a CAP-independent manner at internal ibosome entry sites (IRESes) contained within the mRNA. While extensively studied in picornaviruses, little is known about internal initiation in cellular mRNAs. The boundaries of cellular IRESes have been difficult to define and sequence comparisons show no obvious sequence similarities among cellular IRESes or between cellular and picomaviral IRESes. The Mauro laboratory has analyzed two cellular IRESes contained within the 5' UTRs of the mRNAs that encode Gtx, a homeodomain protein and Rbm3, a cold stress induced protein that is of special interest because it appears to enhance the activity of its own IRES. These studies indicated that some cellular IRE Ses were composed of shorter cis-acting regulatory sequences, some as short as 7-nucleotides, that could function independently to internally initiate translation (IRES-module), or to enhance internal initiation (enhancer element). In addition, a diversity of IRES-modules was selected from a library containing short random nucleotide sequences using a method that was developed in this laboratory. Multiple copies of some naturally-occurring and selected IRESnodules increased internal initiation synergistically. The working hypothesis is that some cellular IRESes are composed of shorter elements that can function independently. The proposed studies will identify naturally-occurring IRES-modules and regulatory elements from cellular mRNAs, while selection studies will attempt to identify sequences with different expression properties. These sequences will be analyzed to determine if they can be categorized, to investigate the rules governing their activity, and to examine if the3 recruit the translation machinery directly by interacting with rRNA or with ribosomal proteins, or indirectly through intermediary proteins. The Rbm3 protein will be studied as a potential example of a trans-acting protein. The proposed studies should provide new insights into our understanding of internal initiation. Inasmuch as several clinically important cellular genes contain IRESes, this increased understanding may allow useful therapeutic manipulations. Moreover, the use of IRE S-modules and enhancers has already resulted in synthetic IRESes that are shorter and more efficient than the large viral IRESes currently used ir dicistronic constructs and should have broad applications in research, gene therapy, and biotechnology.
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