Metabolism, Modes of Action, and Developmental Functions of Small RNAs
Summary: Xuemei Chen uses the weed Arabidopsis thaliana to study noncoding RNAs and stem cells.
Small RNAs as sequence-specific regulators of genes and genomes have emerged as crucial players in numerous biological processes in eukaryotes. Small RNAs fall into three major categories: Piwi-interacting RNAs (piRNAs), found only in animals; and small interfering RNAs (siRNAs) and microRNAs (miRNAs), present in both plants and animals.
The mechanisms underlying the biogenesis and modes of actions of small RNAs are similar in plants and animals in many respects, and studies on plant small RNAs have made fundamental contributions to our knowledge of small RNAs in general. For example, piRNAs have recently been shown to guide DNA methylation at, and the transcriptional gene silencing (TGS) of, transposons; this function is similar to that of endogenous siRNAs in plants. The mechanisms underlying the functions of siRNAs in guiding DNA methylation and TGS have been extensively studied in Arabidopsis. Long noncoding RNAs have also been recognized to play widespread roles as scaffolds or guides in protein complexes, especially those that modify chromatin. In Arabidopsis, long noncoding RNAs have also been implicated in small RNA-guided DNA methylation, a process known as RdDM.
Our lab is taking advantage of the excellent resources and knowledge base in the model species Arabidopsis thaliana to dissect the biogenesis and modes of action of small and long noncoding RNAs. In addition to dissecting the metabolism and functions of noncoding RNAs at the molecular level, we are also studying the biological functions of noncoding RNAs in the context of floral stem cell regulation.
miRNA Biogenesis and Modes of Action
miRNA biogenesis. miRNAs are encoded by MIR genes, which are transcribed by polymerase II (Pol II) into pri-miRNAs that are capped and polyadenylated. The pri-miRNAs undergo at least two consecutive processing steps by DICERLIKE1 to produce the miRNA/miRNA* duplexes that undergo HEN1-mediated methylation. Then the miRNA strand is bound by an ARGONAUTE protein, such as AGO1, the major miRNA effector in Arabidopsis. Our lab has been dissecting this pathway by uncovering the major players involved and revealing their biochemical functions. Our work on HEN1-mediated methylation has revealed the presence of two previously unknown metabolic processes that occur on miRNAs: 3'–5' exonucleolytic degradation and 3' uridylation (the acquisition of a short, U-rich tail).
We have identified a class of small RNA–specific exonucleases composed of SMALL RNA-DEGRADING NUCLEASE1 (SDN1) and its homologs that degrades miRNAs and controls the steady-state levels of miRNAs together with miRNA biogenesis. We have also identified the gene that is responsible for uridylation, and the molecular phenotypes of the mutant in this gene indicate that uridylation promotes miRNA degradation. Work in our lab is focused on determining how the SDN exonucleases and the uridylation enzyme act together to target ARGONAUTE-bound miRNAs for degradation in vivo and how miRNA degradation is regulated in vivo during development or in responses to environmental stresses.
Modes of action of miRNAs. Plant miRNAs are known to guide the precise endonucleolytic cleavage of their target mRNAs to lead to their subsequent degradation. Cleavage is conducted by the endonuclease activity of AGO1. Plant miRNAs are also known to lead to a disproportionate decrease in the protein versus mRNA levels of their target genes, a mode of action that has been referred to as translational inhibition. Although the cleavage-guiding activity of plant miRNAs is well understood, almost nothing is known about how plant miRNAs cause translational inhibition. Animal miRNAs also lead to translational inhibition, but the mechanisms underlying translational inhibition in animals are also poorly understood. Current and future efforts in our lab are directed toward dissecting the mechanisms underlying this mode of action of plant miRNAs and assessing the contribution of this mode of action to the activities of miRNAs.
Long Noncoding RNA Metabolism and Function
Long noncoding RNAs are crucial players in epigenetic regulation: they serve as scaffolds in the assembly of protein complexes or sequence-specific guides in the targeting of chromatin modification complexes to specific regions of the genome. In plants, long noncoding RNAs are implicated in the recruitment of siRNAs to chromatin in the process of RdDM). Long noncoding RNAs are hypothesized and known, respectively, to be produced by Pol IV and Pol V, two plant-specific DNA-dependent RNA polymerases that are critical players in RdDM. Although Pol IV–dependent transcripts have not yet been detected, they are predicted to exist and serve as precursors to endogenous siRNAs at heterochromatic loci. Pol V–dependent transcripts have been detected, and nascent Pol V-dependent transcripts probably play a role in recruiting siRNAs to chromatin. Noncoding RNAs are also known to affect gene expression by recruiting the Polycomb Group (PcG) complex to specific PcG targets.
Although the biological functions of long noncoding RNAs are increasingly being uncovered, the mechanisms underlying their biogenesis, subcellular localization, and degradation are poorly understood. We will use a combination of genomic and genetic approaches to probe the metabolism of long noncoding RNAs, especially those that act in RdDM and in PcG-related functions.
The Temporal Regulation of Floral Stem Cells
All floral organs in a flower are generated by a group of floral stem cells situated at the apex of a floral meristem. Floral stem cells undergo a precise temporal program: they are terminated upon the production of the final floral organ primordia, carpel primordia. Although it may be assumed that the termination of floral stem cells is simply the differentiation of the cells into carpel cells, our studies have shown that this temporal program is distinct from, and coordinated with, the organ identity program. We have identified a number of transcription factors, two miRNAs, a trans-acting siRNA, and PcG as key players in the termination of floral stem cells. Our future studies will aim at unraveling how these factors function at the chromatin, transcriptional, and post-transcriptional levels to bring about a precise temporal program to stem cells.
Grants from the National Institutes of Health, the National Science Foundation, and the United States Department of Agriculture provided partial support for these projects.
As of February 01, 2012