Most human genes produce multiple distinct mRNA and protein isoforms through alternative splicing that may have distinct or even antagonistic biological functions. Alternative splicing is regulated by a set of RNA binding proteins (RBPs) whose functions are important in development and in a number of diseases. Our approach to the long-term goal of understanding the function of RBPs and their roles in alternative splicing is organized around the following specific aims: SA1. To develop a method for determination of the quantitative in vitro RNA binding specificity of a protein at unprecedented depth and to determine the RNA affinity landscape of RBFOX family proteins. SA2. To understand the sequence and RNA structural basis of RNA binding by MBNL and CELF family splicing factors, the extent of cooperativity and competition, and to understand the regulation of distinct subsets of targets in developmental and pathogenic contexts. SA3. To understand the binding affinity landscapes of factors that recognize motifs at the 3'ends of introns and their roles in regulation of alternative 3'splice site (3'SS) choice. We have recently developed a method called HiTS-FLIP, which provides a quantitative description of the in vitro DNA binding affinity landscape of a protein at unprecedented depth. We propose to develop a variation of this method, "HiTS-FLIP-R", to assess RNA binding affinity at a similarly high resolution. The essential idea is to sequence tens of millions of DNA clusters on an Illumina Genome Analyzer 2 (GA2) flow cell, to generate the corresponding RNA sequences by primer extension of anchored DNA adapters, to add a fluorescently tagged RNA binding protein to the flow cell at various concentrations and to image binding to RNA clusters using the GA2's optics. This approach will be applied to key factors involved in development and disease, including RBFOX2 and the myotonic dystrophy (DM) related factors MBNL1 and CELF1, as well as major factors that recognize the 3'splice site and contribute to constitutive and alternative splicing. These data will be used to develop quantitative models that predict the effects on binding and regulation of defined perturbations in the levels of specific RBPs, and the roles of these factors in development and in DM.

Public Health Relevance

This project will provide comprehensive resources, tools and concepts for understanding the binding of proteins to RNA. It will also generate comprehensive binding affinity data for several important RNA binding proteins that function in constitutive splicing, which is required for expression of most human genes, or alternative splicing, including analysis of proteins that play central roles in development and in diseases such as myotonic dystrophy and cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
Zip Code
Lambert, Nicole; Robertson, Alex; Jangi, Mohini et al. (2014) RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins. Mol Cell 54:887-900
Wang, Yang; Xiao, Xinshu; Zhang, Jianming et al. (2013) A complex network of factors with overlapping affinities represses splicing through intronic elements. Nat Struct Mol Biol 20:36-45
Almada, Albert E; Wu, Xuebing; Kriz, Andrea J et al. (2013) Promoter directionality is controlled by U1 snRNP and polyadenylation signals. Nature 499:360-3
Nutiu, Razvan; Friedman, Robin C; Luo, Shujun et al. (2011) Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument. Nat Biotechnol 29:659-64
Spies, Noah; Nielsen, Cydney B; Padgett, Richard A et al. (2009) Biased chromatin signatures around polyadenylation sites and exons. Mol Cell 36:245-54
Xiao, Xinshu; Wang, Zefeng; Jang, Minyoung et al. (2009) Splice site strength-dependent activity and genetic buffering by poly-G runs. Nat Struct Mol Biol 16:1094-100
Wang, Eric T; Sandberg, Rickard; Luo, Shujun et al. (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470-6