Current Research Areas
Alternative pre-mRNA splicing is regulated by trans-acting protein factors binding to cis-acting RNA sequence elements. Current estimates in Drosophila indicate that just over 20% of genes undergo some level of alternative splicing. This number will continue to increase as the transcriptome is more deeply sequenced and more biological samples queried. Some of the protein factors that control alternative and constitutive splicing are conserved between Drosophila and higher eukaryotes, whilst others are not.
PTB
Polypyrimidine tract binding protein (PTB) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that has been implicated in splicing repression in mammals. Drosophila possesses one PTB homolog called hephaestus (heph; dPTB). Hephaestus mutant individuals exhibit male sterility and enlarged testis tip morphology.
With the aim of identifying mRNA transcripts whose splicing is regulated by dPTB, we have carried out RNAi of dPTB in Drosophila Schneider cells. Whole-genome splice-junction microarrays have been employed to detect the splicing events affected by dPTB. Splice junction microarrays were also used to discover splicing events regulated by dPTB in the testis with the hope of better understanding the role of dPTB in testes. These antibodies have also been used to deplete endogenous dPTB ribonucleoprotein complexes (RNPs) from Schneider cell nucleoplasmic fractions to isolate and identify RNA transcripts bound by dPTB. By combining the knowledge of which splice junctions dPTB affects, with information concerning which RNA sites the protein binds to, we are endeavoring to identify dPTB splicing regulatory elements to better understand how location and sequence of these RNA motifs allows dPTB to regulate alternative pre-mRNA splicing in Drosophila. Experiments are currently underway to discover whether any of the mechanisms and specific target RNAs regulatory sites that PTB uses in Drosophila are conserved with mammals.
PSI and hrp48
The third intron (IVS3) of the Drosophila P-element transposase pre-mRNA undergoes tissue-specific alternative pre-mRNA splicing. In somatic cells a repressive splicing complex binds to an exonic splicing silencer containing a pseudo-5’ splice site. Assembly of this RNP complex prevents access by U1 snRNP to the nearby accurate 5’ splice site, which results in retention of the third intron. Biochemical and genetic experiments have previously shown that the hnRNP proteins hrp48 and PSI are part of this silencing complex.
In order to identify novel cellular splicing silencers that use hrp48 and PSI as splicing repressors, we have identified splicing events, using genome-wide approaches, which are co-regulated by both the PSI and hrp48 proteins. RNAi knockdown of hrp48 and PSI in Schneider cells followed by splice junction microarray analysis led to the identification of 12 splicing events, which are affected by both proteins in the same manner, that is to say up or down regulated. In order to evaluate whether the proteins act in combinatorial manner, hrp48 and PSI RNAi knockdowns have been performed separately and together, followed by RT-PCR. Bioinformatic searches for hrp48 and PSI RNA binding site SELEX motifs have been carried out within the RNA regions of the affected pre-mRNA transcripts proximal to the affected splice sites. This information has been used to predict where the two proteins might bind and how the location of these binding sites influences splice site selection. These putative regulatory elements have been tested for activity in mini-gene reporters.
I have a long-standing interest in gene expression at the level of mRNA. My postdoctoral work has focused on mRNA-protein interactions during the nucleur process of splicing whilst my PhD was on 40S ribosomal scanning in the cytoplasm. In the future I hope to gain a better understanding of how the character of mRNPs changed during the lifetime of the mRNP and how the different processes that mRNAs take part in are linked.
Education
Selwyn College, University of Cambridge, UK. PhD in Biochemistry 2006
The Queen’s College, University of Oxford, UK. Masters in Biochemistry 2002
Research Experience
Postdoctoral Research Fellow (2006-present)
Department of Molecular and Cell Biology, University of California Berkeley, CA, USA.
Supervisor: Professor Don Rio
Biochemical characterisation of RNA-protein interactions involved in the regulation of alternative splicing in Drosophila.
PhD student (2002-2006)
Department of Biochemistry, University of Cambridge.
Supervisor: Professor Richard J. Jackson
The ‘biochemical mechanics’ of 40S ribosome scanning.
Undergraduate Research (2001)
Département de Biochimie, Institut National des Sciences Appliquées de Lyon, France.
Supervisor: Dr Catherine Calzada
Summer Research Internship (2001)
Boyce Thompson Institute, Cornell University, NY, USA.
Supervisor: Dr Thomas Brutnell
Summer Research Internship (2000)
MRC Laboratory of Molecular Biology, Cambridge, UK.
Supervisor: Dr Daniela Rhodes
Publications
Aspden, J. L. and Jackson, R. J., Differential effects of nucleotide analogues on scanning-dependent initiation and elongation of mammalian mRNA translation in vitro. RNA 16, 1130-1137 (2010).
Song EJ*, Werner SL*, Neubauer J, Stegmeier F, Aspden J, Rio D, Harper JW, Elledge SJ, Kirschner MW, Rape M. The Prp19-Complex and the Usp4-Sart3 Deubiquitinating Enzyme Control Reversible Ubiquitination at the Spliceosome. Genes & Dev. 24, 1434-1447 (2010).
My research focuses on the P element transposase (TNP) of Drosophila melanogaster, a RNaseH-like protein which is involved in the metal-mediated transposition (i.e. cleavage and subsequent integration) of P-element mobile DNA. Mobile genetic elements or transposons, which have been found in both prokaryotes and eukaryotes, constitute large portions of most eukaryotic genomes (~50% in humans) and have profound effects on gene expression and genome evolution. However, the biochemical mechanisms governing the transposition reaction are not well understood.
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 investigate the role of GTP as a conformational effector in the transposition reaction, I have fused YFP and CFP analogs to the N- and C-terminus of TNP and performed in vitro FRET experiments. 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).
Biochemical characterization of TNP homologs in human and zebrafish: THAP9 proteins are sequence-homologs of TNP which have recently been identified in many vertebrates. Like TNP, THAP proteins are involved in zinc-dependent DNA-binding via an N-terminal THAP domain. However, very little is known about their biological function. Interestingly, TNP stands out among it’s homologs in being the only one known to be involved in active DNA transposition. It also exhibits the highest affinity for it’s corresponding DNA-binding site when compared to all other known THAP proteins; this may explain it’s unique role as a DNA transposase. We are currently conducting cell culture-based transposition assays to check if the THAP proteins (human and zebrafish homologs) can mobilize DNA like TNP, as well as various in-vitro assays to elucidate their role in DNA binding and possible cleavage. Efforts are underway to identify regions of the THAP proteins that are involved in the affinity and specificity of DNA-binding.
Molecular evolution of novel hyperactive P-element transposases: TNP has probably evolved to become a low turn-over enzyme so as not to cause lethal levels of DNA damage in its resident host genome. Hyperactive TNP mutants would allow us to better understand the various steps of transposition and its regulation as well as its evolution as a poor catalyst. Random mutagenic PCR was performed to generate a library of mutant transposases which were screened using a genetic yeast-based selection assay to detect P element excision. A total of 35 residues, which occur throughout the length of the TNP protein, appear to be involved in hyperactivity. Subsequent studies revealed that most of the individual residues mutated in the DNA-binding domain are involved in specificity of DNA-binding. Currently I am further investigating residues in the GTP-binding region as well as acidic residues that may be involved in catalyzing metal-mediated DNA cleavage. This study thus illustrates that like some other transposases, effective transposition by TNP is achieved by a synergistic optimization of various parameters like DNA and co-factor binding and catalysis.
I am studying the biochemical and genetic role of protein complex that binds the Drosophila P element inverted repeats (IRBP Complex). Our data indicates that it regulates the transposition reaction at both the transcriptional level and post-cleavage repair. In fly strains without P elements, the IRBP complex is important for proper DNA repair and fertility. Interestingly, several members of the RNAi machinery are included, suggestive of a novel mechanism to repair DNA breaks.
Generally, I am interested in the regulation of gene expression, both at a gene-specific and genome-wide level. Alternative splicing is a particularly interesting way in which cells can modulate their informational output. The current model for alternative splicing entails a core spliceosome whose location and activity are modulated by extra-spliceosomal splicing factors. My work aims to investigate how these splicing factors do their job, that is, how they bias spliceosomal activity and therefore informational output toward a specific population of mRNA isoforms, using a combination of biochemistry, genetics, and bioinformatics.
We have identified a Drosophila-specific ortholog of the large subunit of the core splicing factor U2AF that we call LS2 (Large Subunit 2). In all organisms from S. pombe to humans, the large subunit of U2AF functions to define 3’ splice sites by recognizing polypyrimidine tracts in the intron and recruiting U2 snRNP. Using RNA SELEX, we have determined that, despite widespread sequence identity between LS2 and U2AF, LS2 binds a G-rich sequence that is very different from the pyrimidine-rich sequence bound by U2AF. Despite this divergence in sequence specificity, we have shown using GST-pulldowns and co-IPs that LS2 retains the ability to interact with the small subunit of U2AF.
Using splice-junction microarrays, we have been able to determine the set of transcripts and splice junctions that are affected by LS2 in S2 cells. These transcripts are enriched for containing the G-rich LS2 recognition motif, for having testes-related gene ontology enrichments, and for being highly expressed in the testes. This is consistent with a large enrichment of LS2 expression in the testes. We have also determined that LS2 is expressed only in differentiated cells in the testes, not in stem cells. We therefore hypothesize that LS2 may play a role in stem cell differentiation in the testes.
Using in vitro and in vivo assays, we have determined that LS2 functions as a splicing repressor. Additionally, we have shown that the LS2 recognition motif is enriched at particular places in alternatively spliced transcripts.
Another project involves investigating a connection between small RNAs and splicing. We have splice junction microarray data showing that Ago2 is involved in the regulation of over 100 splice junctions in the cell. We are currently investigating the mechanism for this regulation.
Finally, we are looking at possible regulation of the splicing factor PSI by specific phosphorylation events. We are working toward identifying the kinase responsible for these events and understanding how these phosphorylations affect the mechanism of PSI action.
Alternative splicing of RNA transcripts is a key eukaryotic mechanism which generates proteomic diversity and regulates gene expression. Additionally, the temporal and spatial control of alternative splicing can give rise to developmental and tissue-specific differentiation, respectively. While splicing is known to be mediated by the spliceosome complex, consisting of five small nuclear ribonucleoproteins (snRNPs) and auxiliary proteins, the mechanisms of splice site selection have yet to be fully characterized.
Splice site selection is controlled by the combinatorial effects of various protein regulators. Facilitating splice site recognition are SR proteins, which recruit U1 and U2 snRNPs via interactions with Arg-Ser repeat domains. Conversely, heterogeneous nuclear ribonucleoproteins (hnRNPs) are often involved in inhibiting splice site recognition by blocking the binding of snRNPs and other regulatory factors. These regulators bind specific RNA sequence motifs known as exonic splicing enhancers (ESEs) and silencers (ESSs).
My project involves examining the interaction of these regulators with ESEs and ESSs in Drosophila. Currently I am utilizing fluorescence-activated cell sorting (FACs) to survey potential motifs involved in enhancement or suppression of exon inclusion in mini-gene reporter constructs. Additionally, interactions between SR proteins, hnRNPs and core splicing components are also being investigated in vitro.
Sima Misra
Montessori Teacher, Berekeley Montessori School, Berkeley, CA
Chris Siebel
Senior Scientist, Genentech, Inc., South San Francisco, CA
Paul Kaufman
Professor, University of Massachusetts Medical Center, Worchester, MA
David Z. Rudner
Associate Professor, Harvard Medical School, Dept of Microbiology & Medical Genetics, Boston, MA
Charles Lee
Chemist, USDA Laboratory, Albany, CA
Melissa Adams
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
Eileen Beall
Research Staff, Dept. of Molecular and Cell Biology, UC Berkeley
Siobhan Roche
Scientist, Trinity College, Dublin, Ireland
Brian T. Weinert
Post-doctoral Researcher, University of Copenhagen, Center for Protein Research, Copenhagen, Denmark
Alejandro (Alex) Sabogal
Post-doctoral Researcher
Douglas Black
Professor, Dept. of Microbiology and Immunology, UCLA, Los Angeles, CA
Yvonne Mul
Scientist, Rotterdam, The Netherlands
Askoa Amarasinghe
Scientist, Stratagene Corp., San Diego, CA
Robin MacDiarmid
Scientist, Horticulture and Food Research Institute, Auckland, NZ
Roland Kanaar
Professor and Director, Department of Cell Biology, Medical Genetic Center, Erasmus University, Rotterdam, The Netherlands
Ottilie Zelenko
Scientist, Vienna, Austria
Emmanuel Labourier
Scientist, Ambion Diagnostics, Inc., Austin, TX
Marco Blanchette
Investigator, Stowers Institute for Biomedical Research, Kansas City, MO
Jenny Kreahling
Postdoctoral Researcher, Moffit Cancer Center, Florida
Li Chen
Scientist, Sigma Corporation, Shanghai, China
Jiro Yasuhara
Patent Office, Tokyo, Japan
Kevin Breger
Medical School, University of Oregon, Portland, OR
Sam Libeu
Medical School
Bosun Min
Postdoctoral Reseracher, Novartis Research Institutes, Emeryville, CA
Ronaldo Panganiban
MSTP Program, Penn State University, College Park, PA
Michael Cho
Reasearch Assistant, San Diego, CA
Sarah Chamanara
Graduate School, Columbia University, New York, NY
Janice Gee
Medical School