cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2ʹ,3ʹ cyclic phosphates and 5ʹ hydroxyl ends

ABSTRACT

Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5′-fragment with a 2′,3′-cyclic phosphate and a 3′-fragment with a 5′-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5′-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3′-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms.

KEYWORDS:

• RtcB ligase
• T4 RNA ligase truncated KQ
• RNA pyrophosphohydrolase (RppH)
• 5ʹ-3ʹ exonuclease Xrn1
• next-generation sequencing

Acknowledgments

We thank Elmar Wahle and Zasha Weinberg for scientific discussions and critical reading of this manuscript and Mario Mörl and his lab for scientific discussions, hosting and supporting the growing Weinberg lab. We also thank Sabine Kleinsteuber and Washington Logroño (Helmholtz Center for Environmental Research, Leipzig) for an introduction to anaerobic cultivation of bacteria, Richard Friedrich for cultivating D. vulgaris and Yuliia Lihanova for conducting co-transcriptional cleavage assays of D. vulgaris HHRs.

Disclosure statement

The authors declare that they have no competing interests.

Author’s contributions

V.J.O. designed and conducted experiments, analyzed experimental data, interpreted experimental and bioinformatics data, wrote the paper, C.G. and J.F. were responsible for sequencing data curation and computational analysis, P.F.S. supervised bioinformatics analysis, J.F. conceived and supervised computational analysis and interpreted sequencing data, C.E.W. conceived the project, designed and analyzed experimental data, interpreted experimental and bioinformatics data and wrote the paper with help from all authors.

Data availability

Raw data of cyPhyRNA-seq corresponding to HighSeq Illumina sequencing of D. vulgaris and to Amplicon sequencing from Ribozyme spike-in into E. coli are uploaded to NCBI:http://www.ncbi.nlm.nih.gov/bioproject/728517Link to genome browser for the D. vulgaris data:http://genome-euro.ucsc.edu/cgi-bin/hgTracks?db=hub_62990_DeVu&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=NC_002937.3%3A1232979%2D1233047&hgsid=269157374_6xbbmG90Mma8tGAAAeBHirYI4aAy

Supplementary material

Supplemental data for this article can be accessed here

Additional information

Funding

This research was supported by a grant of the German Research Foundation(DFG) to C.E.W. (Deutsche ForschungsgemeinschaftLU1889/4) . V.J.O. was supported by a Predoc Award from Leipzig University (jointly given to V.J.O. and C.E.W.) and C.E.W. is also supported by a fellowship of the Peter and Traudl Engelhorn Foundation. Research was also funded in part by the German Federal Ministry for Education and Research (BMBF Bundesministerium für Bildung und Forschung 031A538B, de.NBI/RBC) to P.F.S