Regulation of gene expression by novel plant-specific small nucleolar RNAs

Plant diversity, how plants grow and develop, and how they respond to external stimuli such as attack by pathogens/pests or stress conditions, all depend on the gene content of the plant species and the regulation of expression of the genes. Genes are regulated at many different levels. One important level is where genes are turned on or off or up or down - called transcriptional control. A second level occurs after the gene is transcribed or copied into RNA - called post-transcriptional control. At this level RNAs can be processed in different ways or targeted for destruction or degradation. There are many different mechanisms of post-transcriptional control of which alternative splicing and targeting by small RNA molecules are two of the most important. Alternative splicing is where different portions of a gene transcript are joined in different combinations to generate more than one messenger RNA (mRNA) from a gene. The resultant mRNAs can be translated into proteins with different functions or can be targeted for degradation. Small RNA molecules such as microRNAs (miRNAs) or short-interfering RNAs (siRNAs) can base-pair with target mRNAs and induce their degradation thereby silencing expression of the target gene. This introduces the concept of regulation of mRNAs by the specific interaction via base-pairing of other, usually small, regulatory RNAs. Many essential processes in the cell depend on the activity of small RNAs interacting with other, larger RNA species. For example, the production of ribosomal RNA (rRNA) species found in ribosomes requires small nucleolar RNAs (snoRNAs) to cut the precursor rRNA into specific rRNAs. Messenger RNAs are produced from precursor mRNAs by the process of splicing where introns are recognised and removed. This process depends on small nuclear RNAs for recognition of intron signals and the splicing reaction itself. More recently, hundreds of miRNAs and siRNAs have been identified as regulators of expression by promoting degradation of target mRNAs or interfering with their translation. While different families of small RNAs are thought to interact with and regulate fairly specific sets of target RNAs, examples have been found where small RNAs have picked up the characteristics of other small RNAs such that their range of functionality is not as restricted as previously thought. SnoRNAs are involved in production of rRNA via base-pairing. In animals, some snoRNAs (called 'orphan' snoRNAs) have been found which do not base-pair with rRNAs but instead can base-pair with mRNAs. Some of the human orphan snoRNAs are able to affect alternative splicing of mRNAs showing the evolution of novel functions for snoRNAs. We have found orphan snoRNAs in plants and, in particular, a group of entirely novel, plant-specific snoRNAs. These snoRNAs have complementarity to mRNAs and the major thrust of this proposal is to characterise these new snoRNAs and their unexpected potential to functionally interaction with mRNAs as a new mode of gene regulation in plants. Knowledge of the complexity and subtlety of all aspects of gene regulation is important in understanding how plants grow and survive and in the prediction of responses to changing environments. This mode of regulation is of interest per se in the field of expression control. In addition, because it relies on a stable RNA which can target and base-pair with mRNAs, the potential exists to modify the snoRNA to target other specific genes and processes in a regulated manner. Such information will be an integral part of systems approaches aimed at understanding the interaction networks which regulate gene expression and biological processes.

Small nucleolar RNAs (snoRNAs) are usually involved in interacting with precursor ribosomal RNA to bring about cleavage or nucleotide modifications. It has been known for some time that some expressed snoRNAs do not have sequences to allow targeting of rRNAs but instead have complementarity to mRNA. Recently, human snoRNAs have been shown to be able to interact with and affect alternative splicing of mRNAs. Plants also contain orphan snoRNAs with potential mRNA targets. In particular, we have found three snoRNAs with many novel characteristics which distinguish them from other 'normal' plant snoRNAs and which have a structure not found in other eukaryotes (i.e. plant-specific). We have identified a full knockout mutant for one of these genes which shows a growth phenotype. The overall objective of this proposal is to characterise the novel capped snoRNAs and orphan snoRNAs, to identify their mRNA targets and demonstrate functions for these snoRNAs in regulating gene expression of target mRNAs from protein-coding genes. Initially, we will characterise these snoRNAs in more detail in terms of their expression and protein composition. We will produce over-expression transgenic lines and, if possible, knockouts of the three plant-specific snoRNAs and a family of orphan snoRNAs. We will use microarrays to identify genes whose expression changes in the mutants and over-expressors, and carry out a global alternative splicing analysis using tiling arrays and a new algorithm capable of identifying significant changes in alternative splicing. By combining the experimental data with computationally derived putative mRNA targets, we will select targets for molecular validation and investigation of modes of action. We will use RT-PCR systems and small RNA analyses on mutant and wild-type lines. Finally, we will demonstrate direct interactions between the snoRNAs and mRNA targets by co-expressing mini-genes constructs in plant cells and transfection.