Russ P. Carstens
Assistant Professor, Dept of Medicine

Genetics and Gene Regulation Program

Address

411 Hill Pavilion (office)
480 Hill Pavilion (lab)
380 S. University Avenue
Philadelphia PA 19104-6144

Office tel.: 215 573-1838
Lab tel.: 215 573-1849
Fax: 215 898-0189
E-mail: russcars@mail.med.upenn.edu


Education

Johns Hopkins University, B.A.,1986

Yale University School of Medicine, M.D., 1990

Research Interests

• Study of the molecular mechanisms of alternative splicing.

Key words: Alternative splicing, Post-transcriptional gene regulation, Exons, Introns.

Search PubMed for articles

Description of Research

Project 1: Characterization of post-transcriptional gene regulation by WT1 in podocytes through identification of associated mRNPs.
Products of the Wilm’s tumor gene (WT1) are crucial for kidney development and function. In the adult kidney WT1 is specifically expressed in the podocyte and genetic mutations lead to glomerular disease, highlighting a critical role for this gene in the maintenance of normal renal function. It has been shown that two WT1 splice variants that lead to either inclusion (WT1(+KTS)) or absence (WT1(-KTS)) of three amino acids yield proteins with remarkably divergent functions. The -KTS variant functions as a transcriptional regulator, whereas the + KTS variant interacts with RNA transcripts in the nucleus and cytoplasm and regulates gene expression post-transcriptionally. To identify WT1(+KTS) bound target mRNAs we immunoprecipitated WT1(+KTS) containing mRNPs from cultured podocyte cell lines using a novel epitope tagging system. Using microarray analysis, a set of mRNAs that were highly enriched in WT1(+KTS) immunoprecipitates was identified. Interestingly, the genes encoding these mRNAs included several that have previously been shown to play critical roles in podocyte function. Furthermore, these genes are enriched for those encoding proteins that are expressed at the podocyte slit diaphragm, suggesting that WT1(+KTS) coordinately regulates their expression to maintain proper function of this dynamic structure. Ongoing experiments are underway to determine the functional implications of WT1(+KTS) binding on expression of these target mRNAs. One exciting possibility is that WT1(+KTS) functions to transport and localize these mRNAs to the slit diaphragm and regulate localized translation of slit diaphragm components.

Project 2: Alternative splicing of fibroblast growth factor receptor 2 (FGFR2).
Alternative splicing represents an important mechanism whereby a single gene transcript can give rise to numerous spliced mRNAs, thereby greatly expanding the ribonomic and proteomic diversity that can be obtained from a limited gene number. Despite increasing recognition that the majority of metazoan gene transcripts are subject to alternative splicing, the molecular mechanisms that regulate this process remain poorly understood. We have focused on alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts in which mutually exclusive splicing of two exons, IIIb and IIIc, gives rise to two functionally different receptors, FGFR2-IIIb and FGFR2-IIIc, in epithelial and mesenchymal cells, respectively. These exons encode the C-terminal half of an Ig-like domain in the receptor’s extracellular domain and the resulting receptor isoforms exhibit distinct binding preferences for the FGF family of ligands with important developmental implications. Using a combination of biochemical and genetic approaches we are pursuing the identity of proteins that regulate this splicing choice. A recent advance in the lab has been the development of cell lines expressing fluorescent minigenes that allow detection of the splicing pattern of either exon IIIb or exon IIIc splicing through simultaneous analysis of green and red fluorescence in live cells. Using these “splicing reporters” we are carrying out high throughput, array based cDNA screens to apply functional genomic approaches towards the identification of FGFR2 splicing regulators. Comprehensive identification of the factors that regulate FGFR2 splicing is a first step towards more detailed studies to determine the molecular mechanisms by which they control this and other alternative splicing events.

Project 3: Global analysis of alternative splicing regulation: Experimental identification of regulatory RNA sequence motifs and genomic level characterization of these sequences in alternatively spliced genes.
An important determinant of splicing regulation consists of RNA sequences located in introns that bind regulatory factors and either enhance or silence splicing at adjacent splice sites (Intronic Splicing Enhancers (ISEs) and Intronic Splicing Silencers (ISSs), respectively). These “auxiliary” RNA cis-elements and the proteins that bind them regulate alternative splicing of numerous pre-mRNAs in different cellular milieus; thereby constituting a “cellular code” that determines patterns of alternative splicing. Although specific sequences that function as ISEs and ISSs have been identified in specific regulated gene transcripts, the sequence motifs are largely degenerate and thus a global understanding of sequence motifs that can function to regulate splicing requires elucidation. Using newly developed heterologous minigenes in which the function of an ISE or ISS determines the level of fluorescence in transfected cells, we have screened a library of randomized 20 mers that were inserted downstream of a regulated exon. Using flow cytometry, we have isolated cells that display increased fluorescence due to increased levels of exon inclusion and identified a number of functional ISEs. These experiments are now being scaled up to categorize a comprehensive set of intronic sequence motifs that can regulate splicing of adjacent exons Sequence motifs identified by this approach will then be used in conjunction with bioinformatics approaches to identify global patterns by which they coordinate alternative splicing decisions. We will also pursue the identities of RNA binding proteins that bind to these motifs to expand our understanding of the factors involved in splicing regulation through specific protein-RNA interactions.

Recent Publications

Muh, S. J., Hovhannisyan, R. H., and Carstens, R. P. (2002). A Non-sequence-specific double-stranded RNA structural element regulates splicing of two mutually exclusive exons of fibroblast growth factor receptor 2 (FGFR2). J Biol Chem 277, 50143-50154.

Hovhannisyan, R. H., and Carstens, R. P. (2005). A novel intronic cis element, ISE/ISS-3, regulates rat fibroblast growth factor receptor 2 splicing through activation of an upstream exon and repression of a downstream exon containing a noncanonical branch point sequence. Mol Cell Biol 25, 250-263.

Hovhannisyan, R. H., Warzecha, C. C., and Carstens, R. P. (2006). Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing. Nucleic Acids Res 34, 373-385.

Newman, E. A., Muh, S. J., Hovhannisyan, R. H., Warzecha, C. C., Jones, R. B., McKeehan, W. L., and Carstens, R. P. (2006). Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays. RNA 12, 1129-1141.

Tsai, A., and Carstens, R. P. (2006). An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach. Nat Protoc 1, 2820-2827.

Lab

Rotation Projects

Projects in any of the areas outlined in the Research Description are available and can be discussed in person.

Lab personnel:

Ruben Hovhannisyan-Research Specialist
Behnam Nabet-Research Specialist
Arthur Tsai-Medical fellow
Claude Warzecha-Graduate student