My research program focuses on regulation of alternative splicing in higher eukaryotes, with particular interests in its importance in erythroid development. We are using genome-wide transcriptome analyses to study splicing regulatory networks that extensively modify gene expression during normal development, and are exploring how defects in splicing networks can alter erythroid differentiation. We employ a combination of biochemical and bioinformatics approaches to study the regulatory motifs and splicing factors that control these splicing programs.
Alternative pre-mRNA splicing switches play an important role in regulating gene expression during late erythroid differentiation. 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). More recent experiments show that MBNL1 splicing factor also positively regulates exon 16 splicing.
We are now characterizing the broader erythroid alternative splicing program using exon microarrays (Yamamoto 2009) and RNA-seq analysis of highly purified erythroblasts from mouse and human (manuscript in preparation). These studies have revealed robust alternative splicing in late erythroblasts that is particularly enriched in genes functioning in cell cycle regulation, chromatin structure and function, organelle organization, and RNA processing. Moreover, dozens of these alternative splicing events are regulated in an erythroid differentiation stage-specific manner in concert with the extensive cellular remodeling that occurs in late erythroblasts before enucleation, suggesting an active role for splicing in mediating these events. These studies are in collaboration with Lior Pachter (UC Berkeley), Mohandas Narla (New York Blood Center), and Xiuli An (New York Blood Center).
Our recent studies demonstrate that MBNL1, an RNA binding protein / splicing factor implicated in myotonic dystrophy, plays an important role in erythroid differentiation. MBNL1 knockout mice exhibit differentiation defects accompanied by splicing defects that are currently under further investigation.
Coupling between alternative promoters and downstream alternative splicing. In the protein 4.1R and protein 4.1B genes, alternative promoter/first exon choice is coupled to differential splicing at alternative 3’ splice sites in exon 2, and determines alternative N-terminal structures of the protein. This evolutionarily conserved process proceeds via a two-step “intrasplicing” model that requires a novel splicing element located several kilobases downstream of exon 1A (Parra 2008 and 2012). Future efforts will explore molecular details of the intrasplicing mechanism and investigate how widespread the mechanism might be in regulating other eukaryotic genes.
Rbfox proteins as regulators of tissue-specific alternative splicing. We showed that Rbfox splicing factors are essential components of highly conserved vertebrate alternative splicing programs. This hypothesis is supported by bioinformatics evidence for strong association of proximal intronic Rbfox binding sites with exons expressed in brain-, muscle-, and breast cancer subtype-specific patterns (Brudno 2001, Minovitsky 2005, Das 2007, Gallagher 2011), and biochemical evidence that binding of Rbfox proteins to intronic enhancers downstream of exon 16 promote splicing (Ponthier 2006).
We have also shown that antisense vivo-morpholinos directed against intronic Rbfox splicing enhancer sequences can alter splicing in vivo in mice (Gallagher 2011, Parra 2012).
In collaboration with Gene Yeo (San Diego), we have demonstrated that distal intronic sites, >500nt from regulated alternative exons, contain evolutionarily conserved GCAUG sequences that can bind Rbfox protein and regulate alternative splicing from a distance. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns in endogenous pre-mRNA and demonstrate that a conserved long-range RNA-RNA base-pairing interaction (an RNA-bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene (Lovci, accepted for publication).
Finally, we are now studying the role of Rbfox proteins in mediating muscle-specific splicing in vivo using a zebrafish animal model in collaboration with Sharon Amacher and Thomas Gallagher (Ohio State).
Lovci MT, Ghanem D, Marr H, Arnold J, Gee S, Parra M, Liang TY, Stark T, Gehman LT, Hoon S, Massirer K, Pratt GA, Black DL, Gray J, Conboy JG *, and Yeo GW*. Rbfox proteins regulate alternative mRNA splicing via evolutionarily conserved RNA-bridges. Nat. Struct. Mol. Biol. (accepted for publication, 2013). *co-corresponding authors
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. *co-corresponding authors
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