9752970 Koslowsky This is a POWRE Visiting Professor Award. The phenomenon of mitochondrial RNA editing in kinetoplastid protozoans is the most radical form of post-transcriptional alteration known to date. The editing of messenger RNA transcripts in these organisms can create over half of the mRNA by uridine insertions. Editing generates translatable mRNAs by creating the open reading frames as well as proper initiation and termination signals. The specificity of uridine insertions is provided by a class of RNAs called guide RNAs (gRNAs). These small RNAs are complementary (allowing G:U base pairs) to portions of the fully edited sequence. All of the gRNAs identified to date have defined 5' anchor sequences, guiding sequences and a non-encoded 3' uridine tail. The 5' anchor sequence is required for in vitro editing and is thought to be responsible for selection and binding to the pre-edited mRNA. However, the smallest anchor sequence identified is four nts in length, suggesting that other factors must play a role in specific gRNA selection and use. In addition, almost nothing is known about how the gRNAs are used to direct RNA editing and considerable controversy exists concerning the role the non-encoded U-tail plays in the editing process. The overall objective of this proposal is to define the structures of the gRNA and mRNA as they interact during the editing process. By combining site-specific photoaffinity crosslinking with solution structure probing techniques, the secondary structure of both the gRNA and the mRNA will be analyzed during the initial editing reaction. This information will greatly enhance our understanding of the structure-function relationship involved in the transfer of information from one RNA molecule to another and will help lead to a molecular understanding for the role of gRNAs in the RNA editing process. The ability of small RNAs to dramatically increase the coding capacity of another class of RNA offers strong support for the role of RNA in the o rigins of life. Interactions between the two RNA molecules are fundamental to the editing reaction and the characterization of these interactions in terms of structure-function relationships is crucial for our understanding of this phenomenon. Understanding how the two RNA molecules interact to achieve information transfer may lend considerable insight to the evolution of early replication mechanisms and has substantial implications on our understanding of the storage and regulation of genetic information. This project will be done in collaboration with Dr. H. Ulrich Goringer at the Max-Planck-Institute for Biochemistry in Munich, Germany. Dr. Goringer's laboratory provides a particularly appropriate environment for Dr. Koslowsky to learn the new biophysical approaches she plans to employ in these studies. ***
