Defining molecular and biological functions of Arabidopsis DXO, a protein that removes the non-canonical NAD cap from RNA

The 5’ end of a eukaryotic mRNA generally contains a methyl guanosine cap (m7G cap). In addition to protecting the mRNA from degradation, the cap mediates almost all other aspects of gene expression, including transcription, RNA processing/modification, transport, translation, and gene silencing. A distinctive feature of prokaryotic RNAs is the absence of the 5’-cap. However, in recent years, some bacterial RNAs, particularly a subset of regulatory RNAs and mRNAs, were found to contain a NAD+ moiety at their 5’ ends. Earlier this year, it was reported that yeast and mammalian RNAs also contain the NAD cap. These findings raise intriguing questions about physiological significance and regulatory implications of this nonconventional RNA cap, adding another piece to the complex puzzle of RNA-based regulation of gene expression.

Regulated removal of the RNA cap structure requires de-capping enzymes. Dcp2, a nudix family protein, is the best-known enzyme in eukaryotes that removes the m7G cap from RNA. Very recently, an E. coli nudix protein (NudC) and the yeast and mammalian Rai1/DXO proteins were reported to function as NAD-RNA decapping enzymes. We have found that Arabidopsis DXO (AtDXO) also hydrolyzes NAD from NAD-capped RNAs. We have obtained homozygous atdxo knockout mutants. While the atdxo mutant embryos can develop into viable seeds, the mutant seedlings display multiple developmental defects. The atdxo mutant phenotypes can be fully complemented by transgenic expression of the fission yeast Rai1 gene, indicating functional similarity between the yeast Rai1 and AtDXO.

This proposed study focuses on understanding of molecular and biological functions of the DXO protein and NAD-capped RNAs in Arabidopsis. We will capture NAD-capped RNAs from Arabidopsis using a click chemistry-based method, and the captured RNAs will be sequenced to identify NAD-capped RNAs from both Arabidopsis wildtype and atdxo mutant plants. Profiles of NAD-RNAs and global transcriptomes from wildtype and atdxo will be compared to provide important clues on RNA targets of AtDXO and possible biological pathways mediated by AtDXO. Together with functional analysis of the AtDXO loss-of-function mutations and gain-of-function forms, this study will make a significant contribution to the emerging field of non-conventional RNA capping and decapping.