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

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences


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.

Technical Summary

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.


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Description Small nucleolar RNAs are involved in the processing of ribosomal RNAs for making ribosomes which carry out translation of mRNAs to proteins. In human systems, particular snoRNAs have been shown to also affect alternative splicing of precursor messenger RNAs and therefore the production of specific proteins. This field is still an active area of research but also now includes the impact of, for example, circular RNAs on pre-mRNAs. In the project, we had shown that plant snoRNAs (in Arabidopsis) might also affect alternative splicing. However, this turned out not to be the case and was due (unknown to us) to the heterozygous genetic background of the mutant. The original objectives were therefore changed and new objectives brought in for the remaining time of the project. We produced one main publication on the characterisation of snoRNAs and of the particular snoRNA that we analysed. The new objectives looked at alternative splicing in barley clock genes which has now produced a publication.
Exploitation Route Our findings are generally relevant to the area of alternative splicing in plants which is expanding rapidly. Our results add to overall understanding and to consideration of AS variation among different plant species.
Sectors Agriculture, Food and Drink

Description Alternative splicing of disease-related genes 
Organisation Myongji University
Country Korea, Republic of 
Sector Academic/University 
PI Contribution We have collaborated with Sang Hyon Kim on the alternative splicing of disease resistance genes in Arabidopsis which has emanated from the BBSRC grant.
Collaborator Contribution Sang Hyon Kim worked on the interaction of snoRNAs with pre-mRNAs. He studied potential targets including disease resistance genes in Arabidopsis on his return to Korea. He recently visited my lab on sabbatical.
Impact One publication.
Start Year 2011