Using RNase sequence specificity to refine the identification of RNA-protein binding regions.
Massively parallel pyrosequencing is a high-throughput technology that can sequence hundreds of thousands of DNA/RNA fragments in a single experiment. Combining it with immunoprecipitation-based biochemical assays, such as cross-linking immunoprecipitation (CLIP), provides a genome-wide method to detect the sites at which proteins bind DNA or RNA. In a CLIP-pyrosequencing experiment, the resolutions of the detected protein binding regions are partially determined by the length of the detected RNA fragments (CLIP amplicons) after trimming by RNase digestion. The lengths of these fragments usually range from 50-70 nucleotides. Many genomic regions are marked by multiple RNA fragments. In this paper, we report an empirical approach to refine the localization of protein binding regions by using the distribution pattern of the detected RNA fragments and the sequence specificity of RNase digestion. We present two regions to which multiple amplicons map as examples to demonstrate this approach.
Wang
Xin
X
Division of Biostatistics Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. wang60@iupui.edu
Wang
Guohua
G
Shen
Changyu
C
Li
Lang
L
Wang
Xinguo
X
Mooney
Sean D
SD
Edenberg
Howard J
HJ
Sanford
Jeremy R
JR
Liu
Yunlong
Y
D001665:Binding Sites / Q000235:genetics; D019295:Computational Biology / Q000379:methods; D004268:DNA-Binding Proteins / Q000378:metabolism*; D023281:Genomics / Q000379:methods*; D047468:Immunoprecipitation; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D012313:RNA; D012260:Ribonucleases