My research program focuses on regulation of alternative splicing in higher eukaryotes. 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. With the assistance of wonderful collaborators with expertise in animal model systems, erythroid biology, and bioinformatics approaches, we have studied the regulatory motifs and splicing factors that control these splicing programs. Our previous studies showed that the RBFOX2 binding motif, UGCAUG, is highly enriched downstream of tissue specific alternative exons in brain, in muscle, and in a breast cancer subtype, and that this motif is essential for RBFOX2 enhancer activity in multiple contexts (Brudno 2001, Minovitsky 2005, Das 2007, Lapuk 2009, Lovci 2013). Knockdown of RBFOX proteins in zebrafish alters splicing of numerous genes and results in specific muscle defects including reduced heart rate and in skeletal muscle by complete paralysis (Gallagher 2011).
One major focus of our work is the role of RNA processing during terminal erythropoiesis. Early studies focused on the protein 4.1 gene, which encodes an important structural protein that helps stabilize the red cell membrane. Alternative exon 16 (E16) exhibits a dramatic splicing switch in differentiating erythroblasts, being almost entirely skipped in proerythroblasts but very strongly included in late erythroblasts. We showed that hnRNP A1 is an important silencer of E16 splicing, while RBFOX2 is a major enhancer of splicing (Hou 2002; Ponthier 2006).
More recently, we have been characterizing erythroid RNA processing networks on a more global scale. RNA-seq analysis of FACS-purified human erythroblasts representing the last several cell stages before enucleation has revealed an elaborate and dynamic RNA processing program involving alternative splicing of both exons and introns (Pimentel 2014; Pimentel 2015). Surprisingly, intron retention (IR) is a widespread and dynamic feature of the erythroid transcriptome during late erythropoiesis. Mechanisms that regulate IR are under study. Currently we are also investigating the role(s) of splicing factor MBNL1 during terminal erythropoiesis, focusing on the impact of MBNL1 deficiency in adult bone marrow.
Another of our recurring interests is control of splicing decisions by distant regulatory elements. In the 4.1R gene and its paralog 4.1B, splice site choice at exon 2 (E2) is strictly coupled with promoter choice: transcripts initiated at promoters upstream of an “intraexon” regulatory element always splice via a two-step mechanism to an internal 3’ splice site at E2 (Parra 2008, and Parra 2012). In contrast, transcripts initiated at promoters downstream of the intraexon splice directly to the first 3’ splice site in E2. The intraexon is very distal (>90kb) from the regulated splice site in E2, and is removed in the second step so that it is not represented in mature mRNA. Blocking the regulatory element with antisense vivo-morpholinos (MOs) can re-wire the splicing outcome in vivo (mouse liver).
In the ENAH gene, we identified a distal RBFOX2 splicing enhancer ~1.9kb downstream of alternative exon 11a. The distal enhancer is conserved from birds to mammals, but it functions to enhance splicing only when recruited close to the regulated via an ‘RNA bridge’. Again, an antisense MO that blocks the intronic RBFOX sites can alter splicing in vivo to cause exon skipping (Lovci 2013).
Pimentel H, Parra M, Gee S, Mohandas N, Pachter, N, and Conboy JG. A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis. Nucl. Acids Res., 2015 Nov. 3 pii: GKV1168 [Epub ahead of print] PMID: 26531823.
Pimentel H, Conboy JG, and Pachter L. Keep Me Around: Intron Retention Detection and Analysis. http://arxiv.org/abs/1510.00696, 2015.
Pimentel H, Parra M, Gee SL, Ghanem D, An X, Li J, Mohandas N, Pachter L, and Conboy JG. A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis. Nucl. Acids Res. 42:4031-4042, 2014. PMCID: PMC3973340.
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. 20:1434-1442, 2013. PMCID: PMC3918504. *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. PMCID: PMC3372232.
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. PMCID: PMC3521583. *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: 6033-6039, 2011. PMID: 21156798. PMCID: PMC3057813.
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. PMCID: PMC2911965.
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. PMCID: PMC2665901.
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.
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.
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.