Home Faculty and Research Faculty by Name Donald Rio

Donald Rio

Professor of Genetics, Genomics and Development*
*And Affiliate, Division of Biochemistry and Molecular Biology; Member, Center for Integrative Genetics; C.H. and Annie Li Chair in the Molecular Biology of Diseases

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Research Interests

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 and evolution of organismal complexity in metazoans and leads to significant proteomic diversification. Understanding how pre-mRNA splicing is controlled will be important since at least 40% 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.

Current Projects

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 involve the use of atomic force microscopy (AFM, in collaboration with Carlos Bustamante’s lab) to understand the role that GTP plays in transposition, the detailed reaction pathway and how this cofactor modulates the assembly of transposase on P element DNA. Genetic screens in yeast and bacteria are aimed at identifying hyperactive transposase mutants. 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), the Bloom's DNA helicase and other factors involved in DNA repair. We are investigating the role of these proteins in DNA repair using biochemical, genetic and proteomic approaches.

Biochemistry and genome-wide analysis of alternative pre-mRNA splicing in Drosophila. Using the P element transposon tissue-specific pre-mRNA splicing as a model we showed that regulation of the third P element intron (IVS3) involves RNA binding proteins that recognize an exonic splicing silencer regulatory element in the IVS3 5' exon that results in splicing inhibition. This silencer binds the PSI (P element somatic inhibitor) protein which has four N-terminal KH-type RNA binding domains and a glutamine-rich C-terminal domain that directly interacts with U1 snRNPand modualtes 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. Hrp48 is a member of a class of proteins implicated in silencer function in mammals. We have generated custom alternative splice junction microarrays to monitor genome-wide changes in alternative splicing following inactivation of particular splicing factors using RNAi or specific small molecule inhibitors. Additionally, we are employing biochemical purification, whole genome tiling microarrays and mass spectrometry to identify cellular mRNAs and proteins present in RNP particles containing PSI, hrp48 and other splicing factors. We are also interested in using RNA tagging methods to purify and analyze the protein composition of single transcript RNP particles assembled in vivo. We are interested in whether the incorporation of hnRNP proteins into RNP particles on nascent pre-mRNAs might generate a "code" that specifies 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. Recently, we have shown that this splicing factor effects export of intron-less mRNAs from the nucleus to the cytoplasm.

Selected Publications

Guanosine triphosphate acts as a cofactor to promote assembly of initial P element transposase-DNA synaptic complexes. [Tang, M, Cecconi, C., Kim, H., Bustamante, C. and Rio, D.C. (2005) Genes Dev. 19, 1422-1425]

Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. [Blanchette, M., Green, R.E., Brenner, S.E. and Rio, D.C. (2005) Genes Dev. 19, 1306-1314]

Genome-wide analysis reveals a novel function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs. [Blanchette, M., Labourier, E., Green, R.E., Steven E. Brenner, S.E., and Rio, D.C. (2004) Molecular Cell,14, 775-786]

Distinct contributions of KH domains to substrate binding affinity of Drosophila P-element somatic inhibitor protein. [Chmiel, N.H., Rio, D.C. and Doudna, J.A. (2006) RNA, 12, 283-291]

Interplay between Drosophila Bloom’s helicase and Ku during NHEJ repair of P element-induced DNA breaks. [Min, B., Weinert, B.W., and Rio, D.C. (2004) Proc. Natl. Acad. Sci. USA 101, 8906-8911]

P Element Excision and Repair by Non-Homologous End Joining Occurs in Both G1 and G2 of the Cell Cycle. [Weinert, B.W., Min, B., and Rio, D.C. (2005) DNA Repair, 3, 171-181]

P elements in Drosophila. [D.C. Rio (2002) In Mobile DNA II. ASM Press, pp. 484-518]

Last Updated 2006-08-09