Capture and sequencing of NAD-capped RNA sequences with NAD captureSeq

Abstract

Here we describe a protocol for NAD captureSeq that allows for the identification of nicotinamide-adenine dinucleotide (NAD)-capped RNA sequences in total RNA samples from different organisms. NAD-capped RNA is first chemo-enzymatically biotinylated with high efficiency, permitting selective capture on streptavidin beads. Then, a highly efficient library preparation protocol tailored to immobilized, 5'-modified RNA is applied, with adaptor ligation to the RNA's 3' terminus and reverse transcription (RT) performed on-bead. Then, cDNA is released into solution, tailed, ligated to a second adaptor and PCR-amplified. After next-generation sequencing (NGS) of the DNA library, enriched sequences are identified by comparison with a control sample in which the first step of chemo-enzymatic biotinylation is omitted. Because the downstream protocol does not necessarily rely on NAD-modified but on 'clickable' or biotin-modified RNA, it can be applied to other RNA modifications or RNA-biomolecule interactions. The central part of this protocol can be completed in ∼7 d, excluding preparatory steps, sequencing and bioinformatic analysis.

NAD + -capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated

“…Our NAD captureSeq (workflow depicted in SI Appendix, Fig. S1A) was adapted from the original NAD captureSeq approach developed for bacterial RNAs (13). Total RNAs from seedlings and inflorescences were either treated or not with adenosine diphosphate (ADP)-ribosyl cyclase (ADPRC), which catalyzes the transglycosylation reaction of NAD + .…”

“…Total RNAs were extracted from 12-d-old seedlings and inflorescences by the TRIzol reagent and treated with DNase I (Roche; 1 U/μg RNA at 37°C for 60 min). Isolation of NAD-RNAs was according to the original capture protocol (6,13), with minor modifications as follows: 100-μg portions of total or polysomal RNAs were incubated in 100-μL reactions containing 0.85 μM ADPRC (or left out as a control), 10% (vol/vol) 4-pentyn-1-ol (Sigma), 50 mM Hepes, and 5 mM MgCl 2 (pH 7) at 37°C for 30 min. The reactions were stopped by low-pH phenol/chloroform extraction.…”

The 5’-NAD cap of RNAIII modulates toxin production in Staphylococcus aureus isolates

“…In order to identify those NAD-RNAs from S. aureus, we have made use of NAD captureSeq as 458 described previously (Winz et al, 2017) and applied to 100 µg total RNA from S. aureus ATCC 459 25923 per sample isolated at the late-exponential phase (OD600 = 1.5). A NAD captureSeq variant 460…”

Structural and biochemical studies define Nudt12 as a new class of RNA deNADding enzyme in mammalian cells

“…To directly determine whether Nudt12 contributes to the expression of specific NADcapped RNAs in cells we utilized the NAD captureSeq approach which was successfully used to isolate NAD-capped RNAs in bacteria 2 , yeast 6 and human 7 cells. Briefly, NAD-capped RNAs are identified by the reaction of the 5´ end NAD with ADP ribosylcyclase (ADPRC) to generate an alkyne moiety amendable to click chemistry selective covalent incorporation of biotin and purification of the modified RNA with streptavidin 25 . NAD-captureSeq was carried out with RNAs from HEK293T WT or N12-KO cells.…”