Professor of Genetics, Genomics and Development
*And Affiliate, Division of Biochemistry and Molecular Biology
*Member, Center for Integrative Genomics and Center for RNA Systems Biology
Biochemistry of P element transposase, the mechanism of transposition and activity of P element-related THAP9 genes in humans and zebrafish. The 87kD P element-encoded transposase protein is required to catalyze P element transposition and belongs to a large polynucleotidyl transferase superfamily, that includes RNaseH, RuvC, retroviral integrases, transposases and the argonaute proteins. Biochemical studies using the purified protein revealed that guanosine triphosphate (GTP) is an essential cofactor for the reaction. Current studies involve the use of imaging methods, including atomic force microscopy (AFM), electron microscopy (EM) and total internal reflection fluorescence microscopy (TIRFM) to understand the role(s) that GTP plays in transposition, the detailed reaction pathway and how this cofactor modulates the assembly and activity of transposase on DNA. We are also interested in using X-ray crystallography to study P element transposase bound to DNA, and initially in collaboration with James Berger’s lab, have solved the structure of the N-terminal THAP DNA binding domain (DmTHAP bound to DNA; see figure below). The C2CH zinc-coordinating THAP DNA binding domain is found in many animal genomes in >300 genes. Interestingly, the THAP9 gene in humans and zebrafish shares extensive sequence homology to the Drosophila P element transposase and we have shown that the THAP9 gene encodes an active DNA transposase. We are using genome-wide methods, ChIP-seq, to find locations that THAP9 binds in the human genome.
Structure of DmTHAP-DNA complex. a) Overall structure of the DmTHAP-DNA complex. Experimental electron density map of the DNA (blue mesh) is contoured at 1.5σ. DmTHAP is shown as a ribbon diagram with the βαβ motif highlighted in magenta. Zinc is shown as a green sphere for clarity. b) Base-specific interactions in the major and minor groove. Interacting amino acids are shown as magenta sticks; DNA is shown in surface representation; zinc-coordinating residues are in green.
Biochemistry, genome-wide and systems-level analysis of alternative pre-mRNA splicing and splicing silencer function in Drosophila and humans. The P element transposon pre-mRNA undergoes tissue-specific splicing and we showed that regulation of the third P element intron (IVS3) involves RNA binding proteins (PSI, hrp48, hrp36 and hrp38) that recognize an exonic splicing silencer (ESS) regulatory RNA element in the 5' exon adjacent to IVS3, resulting in splicing inhibition. This RNA silencer element binds the splicing repressor protein PSI (P element somatic inhibitor) protein which directly interacts with U1 snRNP and modulates U1 snRNP binding to specific sites on the pre-mRNA. We are using bioinformatic and genomic (microarray and mRNA-seq) approaches to identify and characterize new splicing silencers that use the hrp48 and PSI factors in cellular genes. 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 a Drosophila paralog of the splicing factor U2 snRNP auxiliary factor (U2AF) that appears to act as a splicing silencer. We are investigating the roles of the microRNA pathway gene, Argonaute-2 (Ago-2), in the nucleus. Using mutants, RNA interference, CLIP-seq and RNA-seq our data indicate that Ago-2 determines genome-wide patterns of alternative pre-mRNA splicing and Polycomb-group (PcG)-mediated transcriptional repression in Drosophila. Finally, as part of the newly formed Center for RNA Systems Biology (Jamie Cate, PI), we embarking on an investigation of transcriptome structure in humans using chemical probing and high-throughput sequencing (SHAPE-seq/DMS-seq) and the role of the RNA chaperone proteins, hnRNA A1 and DDX5 in alternative pre-mRNA splicing decisions and RNA structural alterations, with the goal of linking specific RNA binding or structural features to normal or aberrant splicing patterns.
Center for RNA Systems Biology
Project I. Systems Level Analysis of Alternative Pre-mRNA Splicing: The roles of RNA structure and RNA Chaperone Proteins
PIs: Steven Brenner (UCB), Ming Hammond (UCB), Donald Rio (UCB)
The newly established Center for RNA Systems Biology (CRSB) aims to map the relationship between RNA structural features in a pre-mRNA or mRNA sequences and mRNA fate at a system level. As part of the CRSB, we aim to obtain pre-mRNA splicing and mRNA structural features at a systems level in human cells.
Alternative pre-mRNA splicing is one of the major mechanisms utilized by metazoans to regulate gene expression and to increase the functional diversity of the eukaryotic proteomes. In humans, ~95% of multi-exon genes are alternatively spliced [Pan, 2008] and these RNA processing events have implications for health and disease since disease gene mutations that affect the splicing process result in human genetic disorders [Martin, 2005]. Alternative pre-mRNA splicing is regulated both by RNA-binding proteins that interact with pre-mRNAs and by RNA secondary structures. Recent studies have indicated that RNA secondary structures can play an important, and previously underappreciated, role in the regulation of alternative splicing [Buratti, 2004; Muro AF, 1999; Hiller, 2007]. RNA chaperone proteins are known to alter RNA secondary structure in vitro through RNA-RNA annealing or unwinding and by RNP remodeling [Herschlag 1995, Semrad, 2010]. These RNA chaperones aid in RNA folding, but have also been shown to be involved in splicing and transcription in vivo.
Although much progress has been made in understanding different alternative splicing mechanisms on an individual transcript or gene basis, much remains to be learned including how RNA structure and/or RNA chaperones affects alternative splicing on a global transcriptome-wide level. We aim to systemically link cis-regulatory elements in pre-mRNAs to RNA structural features and protein binding sites that control alternative pre-mRNA splicing in vivo. The RNA binding and chaperone activities of two RNA chaperone proteins, hnRNP A1 and the p68/DDX5 RNA helicase, will be mapped and compared to transcriptome-wide changes in RNA structure using chemical probing information and alternative splicing patterns with RNAi-knockdowns of these factors using high-throughput cDNA sequence analyses.
My research focuses on (1) THAP9, a newly identified vertebrate homolog of the Drosophila P-element transposase (TNP) (2) Structural and biochemical characterization of the P-element transposase (TNP) protein which is involved in the transposition (i.e. cleavage and subsequent integration) of P-element mobile DNA
Newly identified vertebrate homologs of TNP known as THAP9: THAP9 proteins are homologs of TNP which have recently been identified in many vertebrate species including humans and zebrafish. Like TNP, THAP9 proteins are involved in zinc-dependent DNA-binding via an amino-terminal THAP domain. However, very little is known about their exact biological function. Other THAP family members they have been broadly implicated in various cellular processes like cell proliferation, apoptosis, pluripotency and transcription as well as human disease. We have investigated how THAP domains may have evolved, by conserving their overall structural fold but making subtle changes at the individual amino acid level, resulting in diverse DNA-binding specificities and affinities and leading to varied effects on their ability to cleave and mobilize DNA. Also, somewhat unexpectedly, we have demonstrated that THAP9 still retains the catalytic activity to mobilize P transposable elements across species (http://www.sciencemag.org/content/339/6118/446.full). Proteomic and genomic analyses are currently underway to understand the significance of these proteins from a more global perspective. To further investigate the role of THAP9 in zebrafish development we are collaborating with Prof. Sharon Amacher’s lab at the Ohio State University.
Role of oligomerization and nucleotide-dependent conformational changes of TNP in DNA transposition : The TNP tetramer undergoes two-stage binding of P-element DNA to bring about joining of the two P element ends, before donor DNA cleavage and transposition. TNP is unique amongst DNA-transposases in its ability to bind GTP via conserved GTP-binding motifs. GTP appears to allosterically regulate DNA binding by helping the complex find the 2nd TNP-binding site, thus resulting in the assembly of a catalytically active nucleoprotein complex on both transposon ends. The role of GTP hydrolysis in this whole process is not clear: hydrolysis is not required for transposition but may have a yet undetermined role in enzyme recycling and disassembly. To further elucidate the dynamics of this complex process, detailed fluorescence spectroscopic analyses are being carried out in parallel with electron microscopy (with Prof. Eva Nogales) and X-ray crystallography (with Prof. James Berger).
I think the "RNA World Hypothesis" is the most reasonable explanation for the origin of life on this planet. If “life” was once primarily composed of RNA molecules, then the functionality of RNA in biology is undoubtedly under-appreciated. Alternative splicing of mRNA is a great example of how the most subtle of changes in RNA sequence can amplify expression diversity from a single gene.
My current research uses the P-element splicing silencer complex as a model system to investigate mechanisms of alternative-splicing. Earlier studies on the regulation of the Drosophila P-element transposase revealed one of the first examples of tissue-specific alternative splicing. In somatic cells, splicing of the P-element third intron (IVS3) is repressed by formation of a splicing silencer complex on the 5’ exon RNA. This creates an mRNA isoform that retains the intron and translates into a P-element ORF with an early termination codon. Biochemical and genetic experiments have previously demonstrated that the hnRNP proteins PSI and hrp48 silence IVS3 splicing by interacting with U1snRNP near the 5’ splice site. A recent RNAi screen identified 3 additional proteins that are functionally required for efficient silencing of the IVS3 splice site.
Our first aim is to elucidate the molecular mechanism of these proteins in IVS3 splicing silencing. To address this aim I am reconstituting the silencing complex for biochemical and structural analysis. Our second aim is to better understand the role of these proteins in alternative splicing within endogenous genes. To address this aim we are coupling next-generation RNA sequencing with immuno-precipitation to identify the binding sites of these silencing proteins within the transcriptome.
My work combines classical biochemistry and genetics methodologies to understand the interaction between endogenous Drosophila proteins, the P element transposon and transposition. Specifically I have identified the core DNA binding components of the Drosophila Inverted Repeat Binding Protein (IRBP) complex as a heterodimer of two basic leucine zipper proteins, IRBP18 and Xrp1. Together these proteins work in concert to promote efficient DNA repair after P element transposase cleavage. Importantly these proteins function as repair proteins in the absence of P elements. Currently I am employing Chip-exo and RNA-seq to further understand how these proteins work to affect general DNA Repair.
Montessori Teacher, Berekeley Montessori School, Berkeley, CA
Senior Scientist, Genentech, Inc., South San Francisco, CA
Professor, University of Massachusetts Medical Center, Worchester, MA
David Z. Rudner
Associate Professor, Harvard Medical School, Dept of Microbiology & Medical Genetics, Boston, MA
Chemist, USDA Laboratory, Albany, CA
Scientist, Ercole Biotech, Inc., Raleigh/Durham, NC
Linda E. Hammond
Postdoctoral Researcher, Dept. of Molecular Biology and Biochemistry, UC Irvine, Irvine, CA
Carla D. DiGennaro
Instructor, Merritt and Skyline Colleges, Oakland, CA
Jennie L. Warsowe
Genetic Counseling, Cedar Sinai Medical Center, Los Angeles, CA
Research Staff, Dept. of Molecular and Cell Biology, UC Berkeley
Scientist, Trinity College, Dublin, Ireland
Brian T. Weinert
Post-doctoral Researcher, University of Copenhagen, Center for Protein Research, Copenhagen, Denmark
Alejandro (Alex) Sabogal
Professor, Dept. of Microbiology and Immunology, UCLA, Los Angeles, CA
Scientist, Rotterdam, The Netherlands
Scientist, Stratagene Corp., San Diego, CA
Scientist, Horticulture and Food Research Institute, Auckland, NZ
Professor and Director, Department of Cell Biology, Medical Genetic Center, Erasmus University, Rotterdam, The Netherlands
Scientist, Vienna, Austria
Scientist, Ambion Diagnostics, Inc., Austin, TX
Investigator, Stowers Institute for Biomedical Research, Kansas City, MO
Postdoctoral Researcher, Moffit Cancer Center, Florida
Scientist, Sigma Corporation, Shanghai, China
Patent Office, Tokyo, Japan
Medical School, University of Oregon, Portland, OR
Postdoctoral Reseracher, Novartis Research Institutes, Emeryville, CA
MSTP Program, Penn State University, College Park, PA
Reasearch Assistant, San Diego, CA
Graduate School, Columbia University, New York, NY