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Systems Biology of RNA Binding Proteins pp 227–266Cite as

Genetic Variation and RNA Binding Proteins: Tools and Techniques to Detect Functional Polymorphisms

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Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 825))

Abstract

At its most fundamental level the goal of genetics is to connect genotype to phenotype. This question is asked at a basic level evaluating the role of genes and pathways in genetic model organism. Increasingly, this question is being asked in the clinic. Genomes of individuals and populations are being sequenced and compared. The challenge often comes at the stage of analysis. The variant positions are analyzed with the hope of understanding human disease. However after a genome or exome has been sequenced, the researcher is often deluged with hundreds of potentially relevant variations. Traditionally, amino-acid changing mutations were considered the tractable class of disease-causing mutations; however, mutations that disrupt noncoding elements are the subject of growing interest. These noncoding changes are a major avenue of disease (e.g., one in three hereditary disease alleles are predicted to affect splicing). Here, we review some current practices of medical genetics, the basic theory behind biochemical binding and functional assays, and then explore technical advances in how variations that alter RNA protein recognition events are detected and studied. These advances are advances in scale—high-throughput implementations of traditional biochemical assays that are feasible to perform in any molecular biology laboratory. This chapter utilizes a case study approach to illustrate some methods for analyzing polymorphisms. The first characterizes a functional intronic SNP that deletes a high affinity PTB site using traditional low-throughput biochemical and functional assays. From here we demonstrate the utility of high-throughput splicing and spliceosome assembly assays for screening large sets of SNPs and disease alleles for allelic differences in gene expression. Finally we perform three pilot drug screens with small molecules (G418, tetracycline, and valproic acid) that illustrate how compounds that rescue specific instances of differential pre-mRNA processing can be discovered.

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Authors and Affiliations

  1. Center for Computational Molecular Biology, Brown University, Providence, RI, USA

    Rachel Soemedi, Sohini Ramachandran & William G. Fairbrother

  2. Molecular and Cellular Biology and Biochemistry, Brown University, Providence, RI, USA

    Rachel Soemedi, Hugo Vega, Judson M. Belmont & William G. Fairbrother

  3. NIH Undiagnosed Diseases Program, NIH Office of Rare Diseases Research and NHGRI, Bethesda, MD, USA

    Hugo Vega

  4. Ecology and Evolutionary Biology, Brown University, Providence, RI, USA

    Sohini Ramachandran

  5. Center for Biomedical Engineering, Brown University, Providence, RI, USA

    William G. Fairbrother

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  1. Rachel Soemedi
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  2. Hugo Vega
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  3. Judson M. Belmont
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  4. Sohini Ramachandran
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  5. William G. Fairbrother
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Correspondence to William G. Fairbrother .

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  1. UCSD Moores Cancer Center, Institute for Genomic Medicine UCSD Stem Cell Program, La Jolla, California, USA

    Gene W. Yeo

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Soemedi, R., Vega, H., Belmont, J.M., Ramachandran, S., Fairbrother, W.G. (2014). Genetic Variation and RNA Binding Proteins: Tools and Techniques to Detect Functional Polymorphisms. In: Yeo, G. (eds) Systems Biology of RNA Binding Proteins. Advances in Experimental Medicine and Biology, vol 825. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1221-6_7

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