Accurate inference of the full base-pairing structure of RNA by deep mutational scanning and covariation-induced deviation of activity.

Jian Zhan,Junfeng Wang,Peng Xiong,Tongchuan Zhang,Yaoqi Zhou,Zhe Zhang

doi:10.1093/nar/gkz1192

Abstract

Despite the large number of noncoding RNAs in human genome and their roles in many diseases include cancer, we know very little about them due to lack of structural clues. The centerpiece of the structural clues is the full RNA base-pairing structure of secondary and tertiary contacts that can be precisely obtained only from costly and time-consuming 3D structure determination. Here, we performed deep mutational scanning of self-cleaving CPEB3 ribozyme by error-prone PCR and showed that a library of <5 × 104 single-to-triple mutants is sufficient to infer 25 of 26 base pairs including non-nested, nonhelical, and noncanonical base pairs with both sensitivity and precision at 96%. Such accurate inference was further confirmed by a twister ribozyme at 100% precision with only noncanonical base pairs as false negatives. The performance was resulted from analyzing covariation-induced deviation of activity by utilizing both functional and nonfunctional variants for unsupervised classification, followed by Monte Carlo (MC) simulated annealing with mutation-derived scores. Highly accurate inference can also be obtained by combining MC with evolution/direct coupling analysis, R-scape or epistasis analysis. The results highlight the usefulness of deep mutational scanning for high-accuracy structural inference of self-cleaving ribozymes with implications for other structured RNAs that permit high-throughput functional selections.

Highlights

The full base-pairing structure of RNA, resulted from the interplay of secondary and tertiary interactions, serves as a preformed frame for final folding of tertiary structure and is evolutionarily conserved to maintain the structural and functional integrity of an RNA [1]
The deep mutational scanning experiment of cytoplasmic polyadenylation element–binding protein 3 (CPEB3) ribozyme was carried out by random mutations generated from error-prone PCR as shown in Figure 1B with mutation rates varied from 1.73% to 5.62% in three different batches so as to maximize the coverage of double and triple mutations
The combination of covariation-induced deviation of activity (CODA) with a Monte Carlo simulated annealing leads to detection of all Watson– Crick (WC) pairs for twister ribozyme at 100% precision and all WC pairs plus two non-WC pairs for CPEB3 ribozyme at 96% precision, despite that CPEB3 ribozyme has only a small library of

Summary

Introduction

The full base-pairing structure of RNA, resulted from the interplay of secondary and tertiary interactions, serves as a preformed frame for final folding of tertiary structure and is evolutionarily conserved to maintain the structural and functional integrity of an RNA [1]. Full base pairing structures at the single base-pair resolution can only be obtained from highresolution RNA structures determined by X-ray crystallography, nuclear magnetic resonance or cryogenic electron microscopy. These traditional techniques solved only 4112 RNA structures as of 4 November 2018 The most economical method for locating base pairs would be a computational prediction if its accuracy could be assured Such a computational approach is usually referred to as RNA secondary structure prediction many base pairs are associated with tertiary interactions [6].

Methods

Results

Conclusion