NAD tagSeq for transcriptome-wide identification and characterization of NAD+-capped RNAs

Development of NAD tagSeq for genome-wide identification and characterization of NAD+-capped RNAs.

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Some prokaryotic and eukaryotic RNAs have recently been found to contain the NAD moiety as the cap. We describe a method termed NAD tagSeq for transcriptome-wide identification and quantification of NAD-capped RNAs (NAD-RNAs). In this method, NAD-RNAs are labeled with a synthetic RNA and identified by direct RNA sequencing using Oxford nanopore sequencing technology. 

It had long been known that eukaryotic mRNAs generally contain a cap termed m7G cap whereas prokaryotic RNAs do not have the cap. Ten years ago, it was reported that some RNAs in E. coli contain a NAD moiety as the cap. Later, eukaryotic organisms were also found to produce NAD-capped RNAs (NAD-RNAs). These findings indicate a novel mechanism in gene regulation using NAD capping.

We recently developed a method termed NAD tagSeq for transcriptome-wide analysis of NAD-RNAs. This method is based on another method termed NAD CaptureSeq which uses an ADPR cyclase (ADPRC) to replace nicotinamide of NAD with an alkyne followed by copper-catalyzed alkyne-azide cycloaddition (CuAAC) to label NAD-RNAs with biotin. However, in NAD tagSeq, NAD-RNAs are ligated with a synthetic RNAs as a tag, instead of biotin, by using the ADPRC-catalyzed reaction and CuAAC. After tagging, NAD-RNAs are identified by the presence of the RNA tag, through direct RNA sequencing using Oxford nanopore sequencing technology (Figure 1) (

Figure 1 NAD tagSeq links NAD-RNA with tag RNA and then identify NAD-RNAs by direct RNA sequencing with Oxford nanopore sequencing technology. NAD cap is alkynylated with ADPRC, followed by a CuAAC (copper-assisted azide-alkyne cycloaddition) reaction to graft a tag RNA to the 5’ end. A polyadenylation reaction might be needed to ligate a poly(A) tail to RNAs that do not contain a poly(A) tail since only poly(A)-containing RNAs can be sequenced by Oxford nanopore sequencing. The RNAs are identified by direct sequencing by Oxford Nanopore sequencing technology.

NAD tagSeq provides a new method for analyzing NAD+-capped RNAs. The RNA tag serves as a barcode to discriminate the NAD- RNAs from non-NAD-RNAs. NAD tagSeq does not involve PCR amplification which might cause some bias or tedious cDNA library construction and is therefore simpler than NAD captureSeq. NAD tagSeq enables visualization of the overall NAD-RNA structure. The NAD tagSeq method also allow simultaneous measurement of abundance of both NAD-RNAs and non-NAD-RNAs from the same genes.

We believe that NAD tagSeq can be used in identifying and characterizing NAD-RNAs in various organisms. The published protocol of NAD tagSeq describes the detailed procedure that could be followed by other researchers. The availability of the method can facilitate further study to understand functions of NAD-RNAs.

Xiaojian Shao

PhD student, HKBU

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