Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq

Abstract

Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

MONSDA: Modular Organizer of Nextflow and Snakemake driven HTS Data Analysis

“…MONSDA has been successfully applied to a variety of projects, including standard RNA-SEQ and DE analysis [37], Differential transcript usage analysis [38], custom tailored RNA-Seq approaches [29] and a series of projects under review or ongoing. We provide a companion repository https://github.com/jfallmann/MONSDA_companion which will host all configuration files used for analysis of published data and is open to be used as sharing platform all users.…”