Defining the mechanism and function of Arabidopsis mRNA capping enzymes

Messenger RNA (mRNA) in eukaryotic cells typically contains the methylated guanosine (m7G) cap at its 5′ end. The cap is added to a nascent pre-mRNA by three sequential reactions catalyzed by a capping enzyme (CE) and an RNA cap methyltransferase (RNMT). The cap is critical for mRNA stability, pre-mRNA processing, and translation into protein. m7G capping was once regarded as a constitutive housekeeping process for all pre-mRNAs in eukaryotes; however, recent studies support the emerging view that capping is a dynamic process and is modulated in response to stimuli in a gene-specific manner. In addition to the canonical m7G cap, non-canonical RNA caps, such as the NAD cap, have recently been reported. These findings have spurred renewed efforts towards understanding the mechanism underlying mRNA capping and its function(s) in dynamic gene regulation.

Although m7G capping is vital for gene expression and cellular function, there has been no previous report on the enzymology of the capping process or its regulation in plants. Our recent study has led to the discovery of a novel m7G capping mechanism in Arabidopsis: the RNA cap methyltransferase RNMT1 is activated by DXO1, which was previously known to be a decapping enzyme for NAD-capped RNAs. This discovery indicates that plants have evolved a unique and more complex mechanism for controlling the m7G capping process. Our finding also suggests that decapping of NAD-capped RNA and canonical m7G capping are connected in plants. There is still a lack of any report on characterization of the CE in plants. Although other higher eukaryotic organisms generally have a single gene encoding the CE, CE in higher plants is typically coded by multiple genes with additional features, further suggesting more complex control of mRNA capping in plants. This proposal will mainly focus on functional characterization of three Arabidopsis CEs and the interaction of the m7G capping machinery with the RNA polymerase II transcription apparatus. We will also develop methods for genome-wide profiling of capped and uncapped RNA transcripts. This study aims to fill the knowledge gaps around the mRNA capping process and its control in plants and might reveal novel mechanisms and functions of RNA capping that mediate gene expression.