The general interests of our laboratory are the mechanisms and regulation of DNA rearrangements (transposition), DNA repair, and the control of alternative pre-mRNA splicing. We study these processes using the fruit fly, Drosophila melanogaster, as a model system. Our research focuses on the P element family of transposable elements to study the specificity and coordination of these nucleic acid rearrangement reactions and the interplay between P elements and their cellular host. The P element system offers the ability to effectively combine the use of biochemical, genetic, molecular biological and proteomic approaches to study these fundamental aspects of gene regulation. The ability of transposable DNA elements to be mobilized and to cause mutations and chromosomal rearrangements is thought to be important for genome and organismal evolution. In fact, half the human genome is composed of transposons. P element transposition is related to retroviral DNA integration, such as HIV, and to the process by which immunoglobulin and T-cell receptor genes are rearranged in the vertebrate immune system [V(D)J recombination]. P elements use cellular DNA repair pathways to process DNA double-strand breaks generated during P element transposition. A second research interest involves alternative splicing of pre-mRNA, which is an important mechanism for the regulation of gene expression in metazoans and leads to significant proteomic diversification. Understanding how pre-mRNA splicing is controlled will be important since at least 20-30% of the known human and mouse disease gene mutations affect the splicing process. Our studies deal with the interaction of proteins with RNA and DNA as well as the assembly, composition, structure, function and biochemical activities of nucleoprotein complexes.
Biochemistry and regulation of P element transposase, the mechanism of transposition and DNA repair. The 87kD P element-encoded transposase protein is required to catalyze P element transposition. Studies using the purified protein to develop a series of in vitro assays for the different stages of P element transposition revealed that GTP is an essential cofactor for the reaction. Current studies are aimed understanding the role that GTP plays in transposition, the detailed reaction pathway and how modifications can control the biochemical properties of transposase. We are also interested in understanding how Drosophila cells repair DNA strand breaks caused by P element transposase. These broken DNA ends are repaired by a process termed non-homologous end joining (NHEJ). Many of these repair proteins are conserved in evolution and include the ATM family of PI3-related protein kinases (in Drosophila dATM and mei-41) and the Bloom's DNA helicase involved in DNA damage responses. We are investigating whether phosphorylation of transposase might control its activity to be coordinated with the cell cycle, with DNA repair or in response to DNA damage using biochemical, genetic and proteomic approaches.
RNA-protein complexes and the biochemistry of regulated pre-mRNA splicing in Drosophila. P element transposition only occurs in the germline, but not in somatic tissues because of tissue-specific pre-mRNA splicing. The third P element intron (IVS3) is removed only in Drosophila germline tissues to produce the transposase mRNA, but IVS3 is retained in a somatic mRNA that encodes the 66kD repressor protein. This regulation involves RNA binding proteins that recognize a regulatory element in the IVS3 5' exon that results in splicing inhibition. The PSI (P element somatic inhibitor) protein has four N-terminal KH-type RNA binding domains and a glutamine-rich C-terminal domain that directly interacts with U1 snRNP. This interaction promotes U1 snRNP binding to specific sites on the pre-mRNA. Expression of PSI is restricted predominantly to somatic cells and ectopic expression of PSI in the germline inhibits IVS3 splicing. The Drosophila hnRNP protein, hrp48, recognizes the 5' exon regulatory element in vitro and mutations in hrp48 activate IVS3 splicing in somatic cells. We are using cDNA microarrays to identify cellular mRNAs present in RNP particles containing PSI and hrp48. We are interested in whether the incorporation of hnRNP proteins into RNP particles might generate a "code" to how specify how pre-mRNAs are processed. Another project involves U2 snRNP auxiliary factor (U2AF), a member of a family of general splicing factors that contain arginine-serine-rich (R/S) domains. U2AF is a heterodimeric RNA binding protein that recognizes intron polypyrimidine tracts and functions in 3' splice site selection. A new area of investigation involves the relationship between RNA editing by adenosine deaminase acting on RNA (ADAR) and pre-mRNA splicing. In collaboration with Jennifer Doudna's lab, we are attempting to solve X-ray structures of these RNA-protein complexes.
In the News
Three UC Berkeley faculty members - Diana Bautista, Amy Herr and Donald Rio - have been singled out as innovators by the National Institutes of Health and will receive special grants designed to fund "transformative research" that could lead to major advances in medical science.