PUBLICATIONS

(Names of UBM student authors are underlined).

High-Throughput Binding Analysis Determines the Binding Specificity of ASF/SF2 on Alternatively Spliced Human pre-mRNAs

Chang B, Levin J, Thompson WA, Fairbrother WG

Comb Chem High Throughput Screen. 2010 Mar;13(3):242‐52.

PubMed

PMID: 20015017.

Abstract: High-throughput immunoprecipitation (IP) studies of transcription factors and splicing factors have revolutionized the fields of transcription and splicing. Recent location studies on Nova1/2 and Fox2 have identified a set of cellular targets of these splicing factors. One problem with identifying binding sites for splicing factors arises from the transient role of RNA in gene expression. The primary role of most splicing factors is to bind pre-mRNA co-transcriptionally and participate in the extremely rapid process of splice site selection and catalysis. Pre-mRNA is a labile species with a steady state level that is three orders of magnitude (10-3) less abundant than mRNA. As many splicing factors also bind mRNA to some degree, these substrates tend to dominate the output of location studies. Here we present an in-vitro method for screening RNA protein interactions that circumvents these problems. We screen approximately 4000 alternatively spliced exons and the entire Hepatitis C genome for binding of ASF/SF2, the only splicing factor demonstrated to function as an oncogene. From the pre-mRNA sequences returned in this screen we discovered physiologically relevant ASF recognition element motifs. ASF binds two motifs: a C-rich and a purine rich motif. Comparisons with similar data derived from the hnRNP protein PTB reveals little overlap between strong PTB and ASF/SF2 sites. We illustrate how this method could be employed to screen disease alleles with the set of small molecules that have been shown to alter splicing in search for therapies for splicing diseases.

A rapid high-throughput method for mapping ribonucleoproteins (RNPs) on human pre-mRNA

Watkins KH, Stewart A, Fairbrother W.

J Vis Exp. 2009 Dec 2;(34). PMID: 19956082

Abstract: Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor. However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance. We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA. The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA. A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs. Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction. This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.

Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence

Reid DC, Chang BL, Gunderson SI, Alpert L, Thompson WA, Fairbrother WG.

RNA. 2009 Dec;15(12):2385-97. Epub 2009 Oct 27. PMID: 19861426

Abstract: Many splicing factors interact with both mRNA and pre-mRNA. The identification of these interactions has been greatly improved by the development of in vivo cross-linking immunoprecipitation. However, the output carries a strong sampling bias in favor of RNPs that form on more abundant RNA species like mRNA. We have developed a novel in vitro approach for surveying binding on pre-mRNA, without cross-linking or sampling bias. Briefly, this approach entails specifically designed oligonucleotide pools that tile through a pre-mRNA sequence. The pool is then partitioned into bound and unbound fractions, which are quantified by a two-color microarray. We applied this approach to locating splicing factor binding sites in and around ∼4000 exons. We also quantified the effect of secondary structure on binding. The method is validated by the finding that U1snRNP binds at the 5′ splice site (5′ss) with a specificity that is nearly identical to the splice donor motif. In agreement with prior reports, we also show that U1snRNP appears to have some affinity for intronic G triplets that are proximal to the 5′ss. Both U1snRNP and the polypyrimidine tract binding protein (PTB) avoid exonic binding, and the PTB binding map shows increased enrichment at the polypyrimidine tract. For PTB, we confirm polypyrimidine specificity and are also able to identify structural determinants of PTB binding. We detect multiple binding motifs enriched in the PTB bound fraction of oligonucleotides. These motif combinations augment binding in vitro and are also enriched in the vicinity of exons that have been determined to be in vivo targets of PTB.

Simulation of the Human Intracranial Arterial Tree

Leopold Grinberg, Tomer Anor, Elizabeth Cheever, Joseph R. Madsen and George Em Karniadakis

Philosophical Transactions of the Royal Society A, vol. 367, pp. 2371-2386. (2009)

Abstract: High-resolution unsteady 3D flow simulations in large intracranial arterial networks of a healthy subject and a patient with hydrocephalus have been performed. The large size of the computational domains requires the use of thousands of computer processors and solution of the flow equations with about one billion degrees of freedom. We have developed and implemented a two-level domain decomposition method, and a new type of outflow boundary condition to control flow rates at tens of terminal vessels of the arterial network. In the paper we demonstrate the flow patterns in the normal and abnormal intracranial arterial networks using patient-specific data.