Research

5′ RNA capping

5′ capping is important for the function of RNA molecules. It can affect the stability and translation of the RNA and allow the cell to differentiate between its own RNA and foreign uncapped RNA. In recent years, new types of 5′ RNA caps consisting of cellular metabolites has been identified and we are interested in identifying metabolite caps on RNA and characterizing their function.

Metabolite RNA capping

In eukaryotes, mRNA is capped with the canonical m7GpppN cap, which is added early after transcription has initiated. However, in recent years it has become clear that RNAs both in eukaryotes and bacteria can have non-canonical caps. In some cases these caps consist of metabolic nucleotide containing molecules such as NAD + and FAD, which are added to RNA by initiation of transcription.

Examples of metabolite caps on the 5′ end of RNAs.

HCV RNA is 5’FAD capped

We have recently demonstrated that HCV RNA is FAD-capped and that the HCV 5’FAD cap is installed by non-canonical initiation with FAD by the viral RNA-dependent RNA polymerase NS5B. This finding has important implications for HCV biology and suggest that other RNA viruses could be using similar strategies to cap their genomes. Read more about the study in Nature: https://rdcu.be/df8p5

The CapZyme-seq method

CapZyme-seq is a method to identify 5′ metabolite caps on RNA at global scale. The method was first described by the Nickels lab (Vvedenskaya et al., 2018) to detect 5’NAD + capped RNAs. We adopted the method to detection of 5’FAD capped RNA by using a FAD specific Nudix pyrophosphohydrolase from Arabidopsis, AtNUDX23. This enzyme selectively generates 5′ monophosphate on 5’FAD capped RNA, allowing the ligation of a sequencing adapter and the construction of a sequencing library. By comparison to a non-treated control library, the 5′ FAD capped RNAs in the RNA sample can be identified. The use of other enzymes for the CapZyme-seq method allows the detection of other 5’metabolite RNA caps.

AtNUDX23 based global detection of FAD-capped RNAs.