TY - JOUR
T1 - Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS)
AU - Lou, Tzu Fang
AU - Weidmann, Chase A.
AU - Killingsworth, Jordan
AU - Tanaka Hall, Traci M.
AU - Goldstrohm, Aaron C.
AU - Campbell, Zachary T.
N1 - Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2017/4/15
Y1 - 2017/4/15
N2 - RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA.
AB - RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA.
KW - Combinatorial control
KW - NOS
KW - Pumilio
KW - RNA
KW - SELEX
KW - Sequencing
UR - http://www.scopus.com/inward/record.url?scp=85002245014&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85002245014&partnerID=8YFLogxK
U2 - 10.1016/j.ymeth.2016.10.001
DO - 10.1016/j.ymeth.2016.10.001
M3 - Review article
C2 - 27729296
AN - SCOPUS:85002245014
SN - 1046-2023
VL - 118-119
SP - 171
EP - 181
JO - Methods
JF - Methods
ER -