Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS)

Tzu Fang Lou, Chase A. Weidmann, Jordan Killingsworth, Traci M. Tanaka Hall, Aaron C. Goldstrohm, Zachary T. Campbell

Research output: Contribution to journalReview article

13 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)171-181
Number of pages11
JournalMethods
Volume118-119
DOIs
StatePublished - Apr 15 2017

Fingerprint

RNA-Binding Proteins
Throughput
RNA
Proteins
Drosophila Proteins
Genomics
Assays
Experiments
In Vitro Techniques

Keywords

  • Combinatorial control
  • NOS
  • Pumilio
  • RNA
  • SELEX
  • Sequencing

Cite this

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS). / Lou, Tzu Fang; Weidmann, Chase A.; Killingsworth, Jordan; Tanaka Hall, Traci M.; Goldstrohm, Aaron C.; Campbell, Zachary T.

In: Methods, Vol. 118-119, 15.04.2017, p. 171-181.

Research output: Contribution to journalReview article

Lou, Tzu Fang ; Weidmann, Chase A. ; Killingsworth, Jordan ; Tanaka Hall, Traci M. ; Goldstrohm, Aaron C. ; Campbell, Zachary T. / Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS). In: Methods. 2017 ; Vol. 118-119. pp. 171-181.
@article{e955b0d400e44db68ccd6c4c38e9effb,
title = "Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS)",
abstract = "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.",
keywords = "Combinatorial control, NOS, Pumilio, RNA, SELEX, Sequencing",
author = "Lou, {Tzu Fang} and Weidmann, {Chase A.} and Jordan Killingsworth and {Tanaka Hall}, {Traci M.} and Goldstrohm, {Aaron C.} and Campbell, {Zachary T.}",
year = "2017",
month = "4",
day = "15",
doi = "10.1016/j.ymeth.2016.10.001",
language = "English (US)",
volume = "118-119",
pages = "171--181",
journal = "ImmunoMethods",
issn = "1046-2023",
publisher = "Academic Press Inc.",

}

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.

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

VL - 118-119

SP - 171

EP - 181

JO - ImmunoMethods

JF - ImmunoMethods

SN - 1046-2023

ER -