Group I introns are one of the prototypic catalytic RNAs, or ribozymes. Of the more than 100 known group I introns, perhaps ten percent are mobile: they can""""""""home"""""""" as DNA elements to unoccupied alleles of the same gene in which they normally reside. Homing is mediated by a highly specific endonuclease encoded in the intron itself. Most group I introns reside in organellar DNA, but a few have been found in nuclei. All nuclear group I introns are inserted in ribosomal DNA (rDNA). There are only three examples of mobile group I introns in the nucleus, and only two of these have been characterized. They are PpLSU3 and DiSSU1, from the acellular slime molds Physarum polycephalum and Didymium iridis, respectively. Since the introns reside in rRNA, the endonuclease genes embedded in them are transcribed by RNA Polymerase I. Hence these genes are unique in that their presumed messenger RNA apparently is derived from the Pol I transcript. The first major goal of the proposed research is to elucidate how the gene for the I-Ppo endonuclease is expressed from PpLSU3. The work will make use of our previously isolated yeast strains that are resistant tot he lethal effects of the endonuclease. We will determine what sequence elements are needed for successful expression of this protein. For example, is the mRNA for I-Ppo processed from the Pol I transcript, or is it derived from a minor Pol II transcript? Is a functional ribosome needed for expression? Do nucleolar RNA binding proteins play a role in expression? Can other genes artificially placed into this intron also be expressed? DiSSU1 is a self splicing intron with a unique property: Two independent and separable catalytic RNA elements somehow cooperate in the splicing reaction. By sequence the upstream element belongs to the class of group I introns. The downstream element (novel splicing ribozyme, NSR), which by itself can cleave the 3' splice site, appears unrelated to other known catalytic RNAs. The second major goal of the proposed research is to characterize the NSR and to understand how it cooperates with the group I element to orchestrate exon ligation. We will use mutagenesis strategies, including in vitro evolution and selection of revertants, to ask what sequences in the NSR are critical for its function. Can the NSR cleave other RNAs in trans? Can it function in vivo in yeast cells? We will study how the NSR interacts with the upstream group I ribozyme. When expressed as separate molecules, can the two ribozymes form a complex that is competent to ligate exons in trans? If trans splicing works in vitro, we will attempt to set up an in vivo system that will allow selection of mutants in these cooperating ribozymes.
