Very small 19-25 nucleotide RNAs, termed microRNA (miRNA) and silencing RNA (siRNA), have emerged in recent years as key components of major genetic regulatory mechanisms in eukaryotic organisms. While there are similarities between animals and plants both in the small RNA regulatory mechanisms and in their biogenesis, the details differ markedly. In plants, both miRNAs and siRNAs primarily target complementary RNAs for destruction, while in animals miRNAs largely modulate translation. The 21-22 nt miRNAs of plants are important in meristem function, leaf polarity, floral identity and timing, as well as root and vascular development. The longer 24-25 nt siRNAs are involved in viral and pathogen resistance, transgene and transposon silencing, and heterochromatin formation.
MiRNAs are derived from short stem-loop segments of precursors (pri-miRNAs) encoded in the genome either as separate genes or within the transcripts of conventional genes, sometimes in introns. SiRNAs are derived from longer, largely double-stranded RNA molecules that are either produced endogenously or introduced exogenously. Although originally thought to be separate systems, regulatory mechanisms have been described that involve both miRNA and siRNA. The biogenesis of miRNAs in Arabidopsis involves an RNAseIII-family enzyme called Dicerlike1 (DCL1), a dsRNA-binding protein called Hyponastic Leaves1 (HYL1) and an RNA methyl transferase called HUA enchancer1 (HEN1). It may also require a protein encoded by the recently identified SERRATE (SE) gene.
Previous work in this laboratory established that the HYL1 protein is required for processing pri-miRNA to pre-miRNA. The overall goal of this project is to understand the function of the HYL1 protein in miRNA biogenesis through the following specific 3 objectives: 1) characterizing the HYL1 complex, 2) reconstituting miRNA processing in vitro, and 3) investigating the mechanism of pri-miRNA processing and regulation of miRNA genes. Recent progress in the laboratory has allowed an expansion in possible approaches to identifying the proteins involved in miRNA precursor processing. By co-expressing GFP fusion proteins, it was established that DCL1 and HYL1 co-localize to small perinucleolar bodies that are distinct from the similar Cajal bodies that contain the RNA silencing machinery, designated Microprocessor centers. The recent construction of a number of molecular tools puts the project in a good position to reconstitute miRNA processing in vitro, to rapidly analyze the RNA structural requirements for correct miRNA processing and to define the functions of various proteins in pri-miRNA processing. Finally, the regulation of genes encoding the miRNAs themselves will be investigated.
Intellectual merit. This project will enlarge our understanding of an extremely important aspect of gene regulation involving small RNAs in plants. Studies on the HYL1 protein will enhance our basic understanding of miRNA biogenesis in plants, while the regulatory studies will deepen our understanding of the miRNA regulatory hierarchy.
Broader impact. Viral resistance based on small RNA-mediated mechanisms has already proven valuable in agriculture. Greater understanding of small RNA mechanisms is likely to generate beneficial applications, facilitating the development of inducible small RNA-based mechanisms that may allow plants to withstand more extreme environmental conditions than they can at present without compromising their productivity.
Very small 19-25 nucleotide RNAs, termed microRNA (miRNA) and silencing RNA (siRNA), have emerged in recent years as key components of major genetic regulatory mechanisms in eukaryotic organisms. While there are similarities between animals and plants in that both use small RNA to regulate how genes are expressed, the details differ markedly. In plants, both miRNAs and siRNAs primarily target complementary RNAs for destruction, while in animals miRNAs largely the process of protein synthesis. The 21-22 nt miRNAs of plants are important in all aspects of plant development. The longer 24-25 nt siRNAs are involved in viral and pathogen resistance, transgene and transposon silencing, and heterochromatin formation. MiRNAs are derived from short stem-loop segments of precursors (pri-miRNAs) encoded in the genome either as separate genes or within the transcripts of conventional genes, sometimes in introns. SiRNAs are derived from longer, largely double-stranded RNA molecules that are either produced endogenously or introduced exogenously. Although originally thought to be separate systems, regulatory mechanisms have been described that involve both miRNA and siRNA. The biogenesis of miRNAs in Arabidopsis involves an RNAseIII-family enzyme called Dicerlike1 (DCL1), a dsRNA-binding protein called Hyponastic Leaves1 (HYL1) and an RNA methyl transferase called HUA enchancer1 (HEN1). Other RNAseIII-family enzymes called DCL2, -3, and -4 function in the biogenesis of the longer 24-25 nt siRNAs. Our previous work established that the HYL1 protein is required for processing pri-miRNA to pre-miRNA and that it is regulated by the stress hormone abscisic acid (ABA). The work carried out under the present award (MCB 0640186) focused almost exclusively on the defining how the proteins identified by genetic experiments actually cleave the RNA precursors and what structures they recognize in the RNA precursor to cleave it. We established the first plant in miRNA precursor processing system consisting of purified proteins, assessed the contributions of the several component proteins, then went on to define the RNA structural requirements for precursor processing, both in vivo and in vitro. Finally, mutagenesis studies were carried out to identify mutations in proteins that could relieve the mutant phenotype of the original hyl1 mutations and thereby identify additional essential components of miRNA processing. These studies identified a number of such revertants. All of them turned out to affect the gene encoding the DCL1 protein itself, suggesting that the 3-protein system we have studied includes both necessary and sufficient components of the miRNA precursor processing pathway. Further analysis revealed that the mutations increased the rate of the cleavages carried out by the enzyme and confirmed our earlier studies showing that the accessory proteins HYL1 and SE accelerate the rate of miRNA precursor cleavage by the DCL1 enzyme. Taken together, these results significantly advance our understanding of the enzymology and RNA structures necessary for the production of one class of important small regulatory RNAs.
