Year of Award
Doctor of Philosophy (PhD)
Department of Biology.
Arabidopsis thaliana ; Chloroplast DNA ; RNA
Eukaryotic mRNAs generally contain a 5' cap termed as the methyl guanosine cap (m7G cap), which is added to the 5'-end of an elongating transcript soon after the initiation of transcription. This cap plays crucial roles in various steps of gene expression including protection the mRNA from degradation. Recently, non-canonical RNA caps such as the NAD+ cap have been identified at the 5'-end of RNAs from bacteria, yeast and mammals. The role of NAD+ capped RNA (NAD-RNA) is still unclear, and NAD-RNAs have not been reported in plants. A method named NAD tagSeq has been developed in our laboratory for transcriptome-wide identification and characterization of NAD-RNAs. This method is based on specific labeling of NAD-RNAs with a synthetic RNA and followed by single-molecule direct RNA sequencing using the Oxford Nanopore sequencing technology. NAD tagSeq serves as a more accurate approach for the detection and quantification of NAD-RNAs compared to other available methods, as well as providing sequence information of whole transcripts. In the present study, we applied the NAD tagSeq to determine that NAD-RNAs were transcribed from at least a few thousand Arabidopsis genes, mostly protein-coding genes. However, over 60% of the total NAD-RNAs were from < 200 Arabidopsis genes. In general, among NAD-RNA-producing genes, less than ~1% of their transcripts were NAD-capped, but for some genes, over 5% of these transcripts were NAD-RNAs. Top two thousand genes that transcribed relatively high levels of NAD-RNAs were enriched in the photosynthesis, translational processes, and responses to cytokinin and stresses. NAD-RNAs in Arabidopsis shared the similar overall sequence structures and were generally spliced in the similar manner with m7G-RNAs. However, we also identified a small number of un-spliced nascent NAD-RNAs, which supports the notion that the NAD cap added during transcription. The observation that NAD-RNAs were mostly produced from protein-coding genes and spliced like canonical mRNAs suggests that NAD-RNAs likely encode proteins. Arabidopsis protein NUDT7, a nudix family protein, was previously identified to negatively regulate plant immunity, and the mutation of which lead to constitutive activation of the defense response. In an in vitro enzymatic assay, NUDT7 was found to function as a NAD-RNA decapping enzyme. In addition, we showed that the NUDT7 mutation elevated cellular NAD-RNA levels, indicating that NUDT7 might acts as a decapping enzyme in vivo. In order to understand the possible molecular function of NAD-RNAs and the role of NUDT7 in plants through its NAD-RNA decapping ability, we compared the total transcriptomes profiles and NAD-RNAs profiles of the wild-type and the nudt7 mutant to reveal some correlations between NAD-RNA levels and total transcript levels. We found that the NUDT7 mutation elevated the expression of defense-related genes and reduced the expression of many photosynthesis-related and metabolic pathway genes. For many photosynthetic genes, the elevated levels of NAD-RNAs in the nudt7 mutant were associated with reduced total transcript levels from the corresponding genes. This study raises an intriguing possibility that NAD-RNA-mediated gene expression might modulate the cross-talk between chloroplast function and plant immunity.
Includes bibliographical references (pages 89-93)
Zhong, Huan, "Characterization of NAD⁺ -capped RNAs and the function of decapping gene NUDT7 in araiodopis thaliana" (2019). Open Access Theses and Dissertations. 678.
Available for download on Sunday, October 17, 2021