Genome-Wide Approaches for RNA Structure Probing

Abstract

RNA molecules of all types fold into complex secondary and tertiary structures that are important for their function and regulation. Structural and catalytic RNAs such as ribosomal RNA (rRNA) and transfer RNA (tRNA) are central players in protein synthesis, and only function through their proper folding into intricate three-dimensional structures. Studies of messenger RNA (mRNA) regulation have also revealed that structural elements embedded within these RNA species are important for the proper regulation of their total level in the transcriptome. More recently, the discovery of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) has shed light on the importance of RNA structure to genome, transcriptome, and proteome regulation. Due to the relatively small number, high conservation, and importance of structural and catalytic RNAs to all life, much early work in RNA structure analysis mapped out a detailed view of these molecules. Computational and physical methods were used in concert with enzymatic and chemical structure probing to create high-resolution models of these fundamental biological molecules. However, the recent expansion in our knowledge of the importance of RNA structure to coding and regulatory RNAs has left the field in need of faster and scalable methods for high-throughput structural analysis. To address this, nuclease and chemical RNA structure probing methodologies have been adapted for genome-wide analysis. These methods have been deployed to globally characterize thousands of RNA structures in a single experiment. Here, we review these experimental methodologies for high-throughput RNA structure determination and discuss the insights gained from each approach.

High-throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites

“…Because a unique probe sequence (corresponding to a unique taRNA region) is incorporated within each INTERFACE transcript, NGS results not only contain information pertaining to transcript length, indicative of taRNA region accessibility, but also a unique sequence identifier of the corresponding target region. Although this sequence identifier is reminiscent of barcoding approaches that have been used in genome-wide RNA structure probing methods, this system yields specific, “bar-coded” outputs for each interrogated taRNA region, rather than variable RNA-specific fragment outputs 26 . In this way, INTERFACE supports the simultaneous characterization of regional accessibility profiles within any number of RNAs.…”

“…To this end, efforts have recently been made to complement computational predictions with in vivo insights to enhance prediction reliability. For example, in vivo chemical and enzymatic probing methods gauge the level of “protection” or reactivity of individual bases/backbones within a region of interest, and have recently been adapted for high-throughput use 26 , 27 . As these local nucleotide availabilities do not always correlate with regional-level accessibility that more accurately mimics RNA:RNA interactions of regulatory interfaces 28 , efforts have also been placed on methods to quantify regional in vivo RNA hybridization.…”

UPA-seq: prediction of functional lncRNAs using differential sensitivity to UV crosslinking

“…Overall, the simple combination of UV irradiation and phenol-chloroform extraction provides a versatile tool for the prediction of candidate lncRNAs that tightly associate with regulatory proteins. It would be intriguing if we could predict functionality of lncRNAs in the future by combining information obtained by scalable deep sequencing analyses including UPA-seq, eCLIP, and various structure-probing technologies that have been recently developed (for review, see Silverman et al 2016).…”

RNA Regulations and Functions Decoded by Transcriptome-Wide RNA Structure Probing

“…Fortunately, recently we have witnessed the fast development of a new type of approaches, resurging from RNA structure probing analysis with chemicals and enzymes developed as early as 1970s [21] . It has been long known that a wide variety of chemicals and enzymes can react differently with RNA nucleotides in different structure context ( Table 1 ).…”