Abstract
RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin–streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin–SAv roadblocks. We then show that randomly distributed biotin–SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin–SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq.
Highlights
The capacity for RNA to fold into sophisticated structures is integral to its roles in diverse cellular processes including gene expression, macromolecular assembly, and RNA splicing [1,2]
Because RNA folding can occur on a shorter timescale than nucleotide addition by RNA polymerase (RNAP) [3,4,5], a nascent RNA can transition through multiple intermediate structural states as it is synthesized [6]
We recently addressed this technological gap by developing cotranscriptional SHAPE-Seq to measure nascent RNA structures at nucleotide resolution [10]
Summary
The capacity for RNA to fold into sophisticated structures is integral to its roles in diverse cellular processes including gene expression, macromolecular assembly, and RNA splicing [1,2]. The lack of a robust method to directly interrogate RNA structure at nucleotide resolution during transcription has so far limited our ability to fully investigate the fundamental principles of RNA cotranscriptional folding and its impact on generating functional RNA structural states that govern fundamental biological processes. We recently addressed this technological gap by developing cotranscriptional SHAPE-Seq to measure nascent RNA structures at nucleotide resolution [10]. SHAPESeq combines chemical RNA structure probing with high-throughput sequencing to simultaneously characterize the structure of RNAs in a mixture [11,12,13]. SHAPE-Seq reactivities represent the relative flexibility of each nucleotide of an RNA: highly reactive nucleotides tend to be single-stranded, whereas nucleotides with low reactivities tend to be constrained by base-pairing or other intra- or intermolecular interactions [11,16]
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