NADcapPro and circNC: methods for accurate profiling of NAD and non-canonical RNA caps in eukaryotes

Author:

Sharma Sunny,Yang Jun,Favate JohnORCID,Shah PremalORCID,Kiledjian MegerditchORCID

Abstract

AbstractAccurate identification of NAD-capped RNAs is essential for delineating their generation and biological function. Previous transcriptome-wide methods used to classify NAD-capped RNAs in eukaryotes contain inherent limitations that have hindered the accurate identification of NAD caps from eukaryotic RNAs. In this study, we introduce two orthogonal methods to identify NAD-capped RNAs more precisely. The first, NADcapPro, uses copper-free click chemistry and the second is an intramolecular ligation-based RNA circularization, circNC. Together, these methods resolve the limitations of previous methods and allowed us to discover unforeseen features of NAD-capped RNAs in budding yeast. Contrary to previous reports, we find that 1) cellular NAD-RNAs can be full-length and polyadenylated transcripts, 2) transcription start sites for NAD-capped and canonical m7G-capped RNAs can be different, and 3) NAD caps can be added subsequent to transcription initiation. Moreover, we uncovered a dichotomy of NAD-RNAs in translation where they are detected with mitochondrial ribosomes but minimally on cytoplasmic ribosomes indicating their propensity to be translated in mitochondria.

Funder

Foundation for the National Institutes of Health

Publisher

Springer Science and Business Media LLC

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)

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