A Workflow for Identifying Helicase-Dependent RNA Structural Changes in Transcriptional Termination: From DMS Sequencing Analysis to Reporter Assay-Based Validation.

RNA helicases are essential in all aspects of RNA biology where they remodel RNA secondary structures and RNA-protein (RNP) complexes. DEAD-box RNA helicases represent the largest RNA helicase family in eukaryotes. However, identification of biological targets for such helicases has been challenging. Structural probing methods involving treatment of cells with reagents such as SHAPE or dimethyl sulfate (DMS) coupled with sequencing (seq) have now enabled identification of RNA targets for these enzymes. These methods require extensive bioinformatics to derive RNA secondary structure predictions from readouts. Predicted targets must then be experimentally validated to determine the biological relevance of a given RNA structural rearrangement. We have utilized DMS-seq to identify RNA secondary structure targets of the DEAD-box RNA helicase Dbp2 in Saccharomyces cerevisiae and further linked their remodeling to transcription termination efficiency. This chapter outlines a data-analysis workflow for extracting DMS reactivity signals from short-read sequencing data and identifying regions of differential RNA structure between wild-type and dbp2Δ strains. It also describes a reporter assay-based method to assess if predicted structural rearrangements are linked to transcription termination efficiency. These approaches establish an integrated computational framework followed by experimental validation for elucidating helicase-dependent RNA structural dynamics and their functional interplay.