Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing

Abstract

To function in a biological setting, RNA binding proteins (RBPs) have to discriminate between alternative binding sites in RNAs. This discrimination can occur in the ground state of an RNA-protein binding reaction, in its transition state, or in both. The extent by which RBPs discriminate at these reaction states defines RBP specificity landscapes. Here, we describe the HiTS-Kin and HiTS-EQ techniques, which combine kinetic and equilibrium binding experiments with high throughput sequencing to quantitatively assess substrate discrimination for large numbers of substrate variants at ground and transition states of RNA-protein binding reactions. We discuss experimental design, practical considerations and data analysis and outline how a combination of HiTS-Kin and HiTS-EQ allows the mapping of RBP specificity landscapes.

Approaches for measuring the dynamics of RNA–protein interactions

“…The approach is conceptually related to the RNAcompete (Ray et al, ; Ray et al, ) and the RNA bind‐n‐seq methods (Dominguez et al, ; Lambert et al, ), although these techniques do not assess the kinetics of RNA protein interactions. For HiTS‐Kin, RNAs with 7 to 8 randomized nucleotides can currently be used, limited by the sequencing depth (Guenther et al, ; Jankowsky & Harris, ). Bound or processed RNAs are separated from the unbound, or unprocessed RNA species.…”

“…The RNA pool is measured over several time points, which provides the kinetic parameters for all substrate variants. Depending on the reaction setup, obtained kinetic parameters reflect binding kinetics or other reaction steps (Jankowsky & Harris, ).…”

“…Because HiTS‐Kin interrogates the entire sequence space of an RNA pool, sophisticated kinetic specificity models can be developed (Jankowsky & Harris, ; Jankowsky & Harris, ; Niland, Anderson, Jankowsky, & Harris, ). Most current specificity models for RNA–protein interactions are based on equilibrium binding and Position Weight Matrices (PWMs), which assume independent contributions of each nucleotide position to the free binding energy (Guenther et al, ; Zhao & Stormo, ).…”

“…HiTS‐Kin has been applied to several RNA interacting proteins, including C5 from E.coli and RNase H (Kielpinski, Hagedorn, Lindow, & Vinther, ; Lin et al, ). Detailed methodological aspects of the HiTS‐Kin technique have been recently described (Jankowsky & Harris, ). HiTS‐Kin and the corresponding equilibrium technique HiTS‐Eq (High Throughput Equilibrium Binding) can be combined to characterize kinetic landscapes spanning several reaction steps for all sequence variants of a given RNA pool (Jain, Lin, Morgan, Harris, & Tolbert, ; Lin et al, ).…”

A quantitative and predictive model for RNA binding by human Pumilio proteins

“…The above limitations and the importance of regulation by RBPs have sparked a growing interest in developing direct, quantitative genomic-scale approaches for measuring RBP/RNA interactions and affinities. Methods such as MITOMI, HiTS-EQ, HiTS-RAP, RNA Bind-N-Seq and RNA-MaP can provide equilibrium binding constants or apparent affinities Jain et al, 2017;Jankowsky and Harris, 2017;Lambert et al, 2014;Martin et al, 2012;Tome et al, 2014). Of these, RNA-MaP and HiTS-RAP, two related techniques that utilize a v8.0 4 modified sequencing platform and an array of ~10 5 unique immobilized RNA species, eliminate an intermediate capture step that can alter binding occupancies, thereby allowing highly accurate thermodynamic and kinetic binding measurements via fluorescence readout Tome et al, 2014)& vide infra).…”