My research program focuses on regulation of alternative splicing in higher eukaryotes, with particular interests in its importance in erythroid development, in muscle function, and in cancer. The hypothesis that drives much of the work is that splicing regulatory networks extensively modify gene expression during normal development and in disease states including cancer. We employ a combination of biochemical and bioinformatics approaches to study the regulatory motifs and splicing factors that control these splicing programs.
Regulation of alternative pre-mRNA splicing during erythropoiesis. The alternative splicing switch that activates protein 4.1R exon 16 during erythroid differentiation, and ultimately strengthens the red cell membrane, provides an ideal model for studying motifs and factors control tissue-specific splicing. Our studies show that exon 16 splicing is regulated in part by a change in the balance between positive (Fox2) and negative (hnRNP A1) factors during erythroid differentiation (Hou 2002; Ponthier 2006). Future research is aimed at characterizing other factors that contribute to regulation, investigating the mechanism of Fox2 action via the identification of its natural binding partners in late erythroid cells, and testing regulation in vivo using genetic knockin experiments in mice (collaboration with David Bodine, NIH).
We are also characterizing the broader erythroid alternative splicing program. These studies have revealed several new alternative splicing switches during late erythroid differentiation, and we are investigating which splicing factors regulate these events. Collaborators in this work include Tyson Clark (Affymetrix), Amittha Wickrema (U. of Chicago) and Joel Chasis (Berkeley Lab).
Coupling between alternative promoters and downstream alternative splicing. In the protein 4.1R gene, alternative promoter/first exon choice is coupled to differential splicing at alternative 3’ splice sites in exon 2 (Parra 2003), and determines alternative N-terminal structures of the protein. This evolutionarily conserved process (Tan 2005) proceeds via a two-step “intrasplicing” model that requires a novel splicing element located several kilobases downstream of exon 1A (Parra 2008). Future efforts will explore molecular details of the intrasplicing mechanism and investigate how widespread the mechanism might be in regulating other eukaryotic genes.
Fox proteins as regulators of tissue-specific alternative splicing. We hypothesize that the sequence-specific RNA binding proteins Fox1 and Fox2 are essential components of highly conserved vertebrate alternative splicing programs. This hypothesis is supported by bioinformatics evidence for strong association of Fox binding sites with brain- and muscle-specific exons from fish to mammals (Brudo 2001, Minovitsky 2005, Das 2007), and biochemical evidence that binding of Fox proteins to intronic enhancers downstream of exon 16 promote splicing (Ponthier 2006), as well as studies in several other laboratories. We are now studying the role of Fox in mediating muscle-specific splicing in vivo using a zebrafish animal model in collaboration with the Amacher laboratory at UC Berkeley.
Aberrant alternative splicing in cancer. In collaboration with Joe Gray at Berkeley Lab/UC San Francisco, and as part of The Cancer Genome Atlas (TCGA), we are exploring the role of alternative splicing in a variety of human cancers.
Protein 4.1 structure and function. In collaboration with Mohan Narla (New York Blood Center), we have long been interested in function of the prototypical protein 4.1R gene and its paralogs in other tissues. We have generated mouse knockouts for 4.1R, 4.1G, and 4.1N in order to study their physiological functions in erythroid and other tissues.
Parra MK, Gallagher TL, Amacher SL, Mohandas N, and Conboy JG. Deep intron elements mediate nested splicing events at consecutive AG-dinucleotides to regulate alternative 3’ splice site choice in vertebrate 4.1 genes. Mol. Cell. Biol. 32:2044-53, 2012.
Gallagher TL, Arribere JA, Geurts PA, Dill KK, Marr HL, Adkar SS, Garnett AT, Amacher SL and Conboy JG. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle function. Developmental Biology 359: 251-261, 2011.
Parra MK, Gee S, Mohandas N, and Conboy JG. Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice. J. Biol. Chem. 286:6033-9, 2011.
Lapuk A, Marr, H, Jakkula L, Pedro H, Bhattacharya S, Purdom E, Hu Z, Simpson K, Pachter L, Durinck S, Wang N, Parvin B, Fontenay G, Speed T, Garbe J, Stampfer M, Bayandorian H, Dorton S, Clark TA, Schweitzer A, Wyrobek A, Feiler H, Spellman P, Conboy J and Gray JW. Exon-level microarray analyses identify alternative splicing programs in breast cancer. Mol Cancer Res. 8:961-74, 2010.
Yamamoto ML, Clark TA, Gee SL, Kang J-A, Schweitzer AC, Wickrema A, and Conboy JG. Alternative Pre-mRNA Splicing Switches Modulate Gene Expression in Late Erythropoiesis. Blood 113:3363-70, 2009.
Purdom E, Simpson KM, Robinson MD, Conboy JG, Lapuk AV, Speed TP. FIRMA: a method for detection of alternative splicing from exon array data. Bioinformatics 24:1707-14, 2008.
Parra MK, Tan JS, Mohandas N and Conboy JG: Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene. EMBO J. 27:122-131, 2008.
Das D, Clark TA, Schweitzer A, Yamamoto M, Marr H, Arribere J, Minovitsky S, Poliakov A, Dubchak I, Blume JE, and Conboy JG. A Correlation with Expression Approach to Identify cis-Regulatory Elements for Tissue-Specific Alternative Splicing. Nucl. Acids Res. 35: 4845-57, 2007.
Ponthier JL, Schluepen C, Chen W, Lersch RA, Gee SL, Hou VC, Lo AJ, Short SA, Chasis JA, Winkelmann JC, Conboy JG. Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16. J. Biol. Chem. 281:12468-74, 2006.
Tan JS, Mohandas N, and Conboy JG: High frequency of alternative first exons in erythroid genes suggests a critical role in regulating gene function. Blood 107: 2557-61, 2006.
Tan JS, Mohandas N, and Conboy JG: Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes. Genomics 86:701-707, 2005.
Minovitsky S, Gee SL, Schockrpur S, Dubchak I and Conboy JG: The splicing regulatory element, ugcaug, is phylogenetically and spatially conserved in introns that flank tissue-specific alternative exons. Nucl. Acids Res. 33: 714-724, 2005.
Parra MK, Gee SL, Koury MJ, Mohandas N, Conboy JG: Alternative first exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini Blood 101: 4164-4171, 2003.
Hou, VC, Lersch R, Gee SL, Wu M, Turck CW, Koury M, Krainer AR, Mayeda A, Conboy JG. Decrease in hnRNP A/B expression during erythropoiesis mediates a pre-mRNA splicing switch. EMBO J. 21: 6195-6204, 2002.
Brudno M, Gelfand MS, Spengler S, Zorn M, Dubchak I, Conboy JG: Computational analysis of candidate intron regulatory elements for tissue-specific alternative pre-mRNA splicing. Nucl. Acids. Res. 29: 2338-2348, 2001