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.

Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice

“…SEQRS was conducted as described with minor modifications on PABPC1 62 . The initial RNA library was generated from transcription of 1 μg of double-stranded DNA using the AmpliScribe T7-Flash Transcription Kit (Epicentre).…”

“…The ssDNA product was used as a template for 25 cycles of PCR using a 50 μL GoTaq reaction (Promega). Sequencing data were processed as described 62 . Sequence logos corresponding to consensus binding motifs were generated by weblogo from the top 300 most enriched sequences.…”

Linking RNA Sequence, Structure, and Function on Massively Parallel High-Throughput Sequencers

“…In vitro approaches can solve a number of these uncertainties, and medium-throughput approaches for quantitative measurement have been enabled by microfluidic methods (Martin et al 2012). Higher-throughput measurements of relative RBP affinity have been achieved with sequencing-enrichment approaches like RNAcompete, RNA-bind-and-seq, RNA SELEX, HiTS-Kin, and HiTS-Eq (Ellington and Szostak 1990;Ray et al 2009;Lambert et al 2014;Jankowsky and Harris 2017;Lou et al 2017). These methods have enabled measurements of relative RBP affinity across randomized libraries; however, they often require immense sequencing resources for readout and the enrichment readout is not as generalizable as direct measurements of biophysical parameters.…”

Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

“…However, prior in vitro determination of human PUM sequence preferences have involved only one round of selection [51] or a selected subset of possible sequences [52]. Thus, to compare the binding specificity of the PUM-HD of the human PUM1 and human PUM2 proteins we applied in vitro selection and high-throughput sequencing of R NA and s equence specificity landscapes (SEQRS) to purified PUM-HDs of each protein [53]. Similar to s ystematic e volution of ligands by ex ponential enrichment (SELEX) [54], SEQRS allows for the determination of an RNA-binding protein’s sequence specificity by selecting for RNAs that interact with the RBP out of a pool of random 20mers generated by T7 transcription of a synthesized DNA library.…”