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The role of natural antisense transcripts in plant gene regulation

Reference	BB/D015774/1
Principal Investigator / Supervisor	Professor Peter Meyer
Co-Investigators / Co-Supervisors
Institution	University of Leeds
Department	Inst of Integrative & Comparative Biolog
Funding type	Research
Value (£)	299,899
Status	Completed
Type	Research Grant
Start date	01/10/2006
End date	30/09/2009
Duration	36 months

Abstract

Plant genomes contain a surprisingly large number of antisense transcripts that partly overlap with a corresponding sense transcript. Among all genes that encode a protein, we have identified 956 convergently overlapping gene pairs (COPs) in the Arabidopsis genome. Expression of an antisense gene can theoretically influence sense gene transcription (transcriptional interference), or it could influence stability, processing or export of the sense transcript. Our analysis of the 956 COPs revealed a strong bias in favour of sense genes to be alternatively spliced and/or alternatively polyadenylated. This suggests a role for antisense transcripts in sense transcript processing for certain COPs. For another subgroup of COPs, we find antagonistic expression profiles for the sense and antisense gene, which may indicate transcriptional interference. Another group overrepresented among COPs, consists of sense genes with a circadian clock expression profile. For COPs representing these different subgroups, we propose to test if and how antisense transcription influences sense transcription or transcript modification. The extensive microarray databases and insertion collection that are available for Arabidopsis genes helped us to select COPs with significant changes in sense/antisense transcript ratios in different tissues, for which we can obtain mutants with insertions that only affect the antisense transcript. We will test several COPs for each subgroup, and examine the effects of changes in sense/antisense ratios in wildtype plants, and of inhibition of antisense transcription in mutants. Transcripts and transcript variants will be quantified by RT-PCR using specific primers for individual splicing and polyadenylation variants. As dsRNA formation can influence transcript export, we will also examine potential changes in nuclear and cytoplasmic distribution of sense transcript variants in the presence and absence of antisense transcript.

Summary

In higher organisms, genetic information that is responsible for the production of a protein, is transcribed into an RNA molecule in the nucleus, which is then processed into a messenger RNA and exported into the cytoplasm for translation into an amino acid sequence. Protein-encoding genes only constitute a minority of the genomes of most organisms. In theory, there should therefore be sufficient space for individual genes to be separated from each other on the chromosomes, to ensure that transcription processes do not interfere with each other. When we analysed the distribution of protein-encoding genes in the model plant Arabidopsis thaliana, we found, to our surprise, that a considerable number of genes (a total of 956) partially overlap with other genes, in a sense-antisense arrangement. We named these loci Convergently Overlapping Gene pairs (COPs). This contrasts historical arguments that the convergent arrangement of overlapping genes is detrimental and has been preferentially removed during evolution. When we examined expression data from about 1,400 individual experiments, we found that both the sense and the overlapping antisense transcripts of the 956 COPs are often present in the same tissue types. This implies that the two transcripts could align to form double-stranded RNA (dsRNA) molecules. We know that dsRNA molecules can be recognised by a degradation mechanism, termed RNA interference (RNAi), which causes the destruction of both transcripts. Our analysis of the distribution of sense and antisense transcript, which we have recently published, shows, however, no indication for a specific degradation of COPs transcript. We know from animal systems that antisense transcripts can fulfil alternative roles than forming dsRNAs for the RNAi system. In plants, however, no such alternative pathways have been described. We therefore would like to find out, which alternative pathways plants have, for which antisense transcripts are required. Our analysis of theorganisation and expression of the 956 COPs helped us to make an educated guess to postulate two alternative functions for antisense transcripts. (i) Among COPs, we find a much higher than expected proportion of genes that have more than one transcript, either because different regions are removed from the original RNA copy (alternative splice variants) or because the transcripts differ in length (alternative polyadenylation variants). This implies that antisense transcripts are required to modify sense transcript processing. (ii) We also frequently find that, while both the sense and antisense genes are transcribed, they often behave antagonistically: In many tissue types, one transcript type is high and the other low. For COPs where a particular stress treatment increases sense transcript levels, the antisense transcript level is reduced. Very soon, however, antisense transcript levels increase and sense transcript levels are reduced. A similar antagonistic pattern can be seen for COPs genes whose transcript levels change periodically during the day (circadian clock genes). These observations suggests a 'trancription interference' model, where transcription of one gene affects transcription of the other, which may be used to reset transcript levels after induction. We want to test if and how antisense transcripts can influence the production or composition of sense transcripts. For this we have selected individual COPs as potential model genes for each of the two mechanisms that we propose. We use Arabidopsis, because for each of our model COPs, there is a mutant line, with an insertion in the antisense gene. These lines allow us to assay if and how sense transcription or transcript processing is altered if the antisense gene in inactive. We think that our work will help to widen the current perception of gene expression, which focuses on transcription and transcript stability, but does not consider the regulatory effects from antisense transcripts.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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