Faculty and Research
Faculty by Name
Donald Rio
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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
Research Interests
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The general interest of our laboratory is nucleic acid transactions, and specifically, 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 these nucleic acid rearrangement reactions and the interplay between P elements and their cellular host. Recent genome sequencing has revealed that P element-related genes are present in vertebrates, including humans and zebrafish. The P element system offers the ability to effectively combine the use of biochemical, genetic, molecular biological and proteomic approaches to study fundamental aspects of gene regulation, genome rearrangements and DNA repair. 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 these complex nucleoprotein machines.
Current Projects
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Biochemistry and regulation of P element transposase, the mechanism of transposition, P element P element-related genes in humans and zebrafish, 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 are identifying hyperactive transposase mutants. We are using genetic and biochemical approaches to define the domain organization of P element transposase and, in collaboration with James Berger’s lab are carrying out X-ray structure determination of the N-terminal THAP DNA binding domain. The C2CH THAP DNA binding domain is found in many organisms, and the THAP9 gene in humans and zebrafish shares extensive sequence homology to P element transposase. We are also interested in understanding how Drosophila DNA repair machinery is deployed to sites of DNA strand breaks caused by P element transposase. We are investigating biochemically and genetically proteins involved in this DNA repair process.
Biochemistry and genome-wide analysis of alternative pre-mRNA splicing and splicing silencer function 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 (ESS) regulatory element in the IVS3 5' exon that results in splicing inhibition. This silencer element 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 snRNP and modulates U1 snRNP binding to specific sites on the pre-mRNA. We are using bioinformatic and genomic approaches to identify and characterize other splicing silencers that use the hrp48 and PSI factors in cellular genes. We have designed and used 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 reconstitution and 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 using RNA tagging methods to purify and analyze the protein composition of RNP and splicing silencer complexes assembled in vitro and 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) and alternate U2AF-like subunits in Drosophila. U2AF is a heterodimeric RNA binding protein that recognizes intron polypyrimidine tracts and functions in 3' splice site selection. We have shown that this splicing factor effects export of intronless mRNAs from the nucleus to the cytoplasm. Finally, we are investigating whether small microRNAs can affect alternative pre-mRNA splicing in Drosophila.
Selected Publications
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Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome. [Sharma, S., Kohlstaedt, L.A., Damianov, A., Rio, D.C. and Black, D.L. (2008) Nat. Struct. Mol. Biol. 15, 183-91]
A regulator of mutually exclusive splicing fidelity in Dscam. [Olson, S., Blanchette, M., Park, J., Saava, Y., Yeo, G.W., Yeakley, J.M., Rio, D.C. and Graveley, B.R. (2007) Nat. Struct. Mol. Biol., 14, 1134-1140]
Analysis of P element transposase protein-DNA interactions during the early stages of transposition. [Tang, M, Cecconi, C., Kim, H., Bustamante, C. and Rio, D.C. (2007) J. Biol. Chem., 282, 29002-29012]
DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom’s syndrome helicase. [Weinert, B.W. and Rio, D.C. (2007) Nuc. Acids Res., 35, 1376]
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]
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]
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]
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]
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]
Last Updated 2008-08-16