Research area 1. Small RNAs

Small RNAs are sequence-specific regulators of gene expression and genome stability. Depending on their sizes and associated Argonaute proteins, small RNAs cause transcriptional or post-transcriptional gene silencing. Our lab mainly studies miRNAs and endogenous siRNAs that cause posttranscriptional gene silencing. Two main areas of study at present are as follows.

1. Small RNAs and the translation machinery

Our lab was among the first to show that translation repression is a mode of action of plant miRNAs [1]. Our work pinpointed the rough ER as the site of action of miRNAs in translation repression [2], but how miRNAs cause translation repression is still poorly understood and is being actively investigated in the lab.

While miRNAs regulate the translation of target mRNAs, the translation machinery may also affect the activities of miRNAs. We found that several “non-coding” RNAs that are targeted by miRNAs to produce secondary siRNAs (known as phased siRNAs or phasiRNAs) are associated with membrane-bound polysomes [3]. The ribosomes are positioned in a manner that strongly implicates a role of ribosomes in the production of phasiRNAs. We are investigating the interactions between small RNA metabolism and translation.

2. The cell-to-cell movement of miRNAs

It has long been known that RNA silencing via siRNAs moves cell-to-cell as well as systemically in plants. Historically, miRNAs were thought to act cell autonomously. However, increasing evidence in recent years points to the cell-to-cell and systemic movement of miRNAs in various plants and leads to the recognition of miRNAs as informational molecules that coordinate tissues/organs in developmental patterning or stress responses [4].

While the non-cell autonomy of miRNAs is increasingly recognized, almost nothing is known about the mechanisms that govern/regulate the non-cell autonomy of miRNAs. Our on-going research is directed towards understanding such mechanisms. We are using the Arabidopsis root, in which miR165/6 is produced in the endodermis but acts in xylem specification in the stele, as one of the models. We have isolated, and are characterizing, mutants that are defective in the non-cell autonomous activities of miRNAs.

Research area 2. Reversible RNA modifications

The textbook version of the life of an mRNA is that an m7G cap is added to the mRNA soon after transcription initiation, and the cap promotes splicing, polyadenylation, nuclear export, stability, and translation of the RNA. In recent years, the redox agent and metabolite, nicotinamide adenine diphosphate (NAD+), has emerged as an RNA cap in bacteria, yeast, and human cells. Work from my group and that of my collaborator Yiji Xia’s group showed that NAD+-capped mRNAs are widespread in Arabidopsis thaliana [5, 6]. NAD+ is incorporated into RNA as the first nucleotide by RNA polymerases during transcription, and can be removed by two types of decapping enzymes, the Nudix family found in bacteria and eukaryotes, and DXO found in eukaryotes.

The discovery of a new RNA cap raises numerous questions, the answers to which may refine or alter the current paradigms of RNA metabolism. On the mechanistic side, what determines the incorporation of the NAD+ cap in an mRNA? What does the NAD+ cap confer to an mRNA? How is NAD+ capping/decapping regulated? How does NAD+ capping/decapping interface with other processes an mRNA undergoes, such as export, degradation and translation? On the biological side, what biological processes are regulated by NAD+ capping/decapping? As NAD+ serves critical functions in cellular redox and energy homeostasis, it is possible that RNA NAD+ capping/decapping is both regulated by and impacts cellular redox and metabolic homeostasis.

We are addressing some of the questions by characterizing mutants in the decapping enzymes and by searching for the “reader” of the cap through biochemical approaches.

We are also combining biochemical and analytical approaches to identify other RNA caps. We suspect that cellular metabolism can influence gene expression through metabolites serving as non-conventional RNA caps. 

References

1. Chen, X. (2004). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303, 2022-2025.
2. Li, S., Liu, L., Zhuang, X., Yu, Y., Liu, X., Cui, X., Ji, L., Pan, Z., Cao, X., Mo, B., et al. (2013). MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153, 562-574.
3. Li, S., Le, B., Ma, X., Li, S., You, C., Yu, Y., Zhang, B., Liu, L., Gao, L., Shi, T., et al. (2016). Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis. Elife 5.
4. Liu, L., and Chen, X. (2018). Intercellular and systemic trafficking of RNAs in plants. Nat Plants 4, 869-878.
5. Wang, Y., Li, S., Zhao, Y., You, C., Le, B., Gong, Z., Mo, B., Xia, Y., and Chen, X. (2019). NAD(+)-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated. Proc Natl Acad Sci U S A 116, 12094-12102.
6. Zhang, H., Zhong, H., Zhang, S., Shao, X., Ni, M., Cai, Z., Chen, X., and Xia, Y. (2019). NAD tagSeq reveals that NAD(+)-capped RNAs are mostly produced from a large number of protein-coding genes in Arabidopsis. Proc Natl Acad Sci U S A 116, 12072-12077.