The role of natural antisense transcripts in plant gene regulation

在高等生物中，负责产生蛋白质的遗传信息被转录成细胞核中的RNA分子，然后被加工成信使RNA，并输出到细胞质中，翻译成氨基酸序列。编码蛋白质的基因只占大多数生物基因组的一小部分。因此，理论上应该有足够的空间让单个基因在染色体上相互分离，以确保转录过程不会相互干扰。当我们分析蛋白质编码基因在模式植物拟南芥中的分布时，我们惊讶地发现，相当多的基因(总共956个)以正义-反义排列的方式与其他基因部分重叠。我们将这些基因座命名为可收敛重叠的基因对(COPS)。这与历史上的观点形成了鲜明对比，历史观点认为，重叠基因的聚合排列是有害的，并且在进化过程中优先被移除。当我们检查了大约1400个单独实验的表达数据时，我们发现956个COP的正义和重叠的反义转录本通常存在于相同的组织类型中。这意味着这两个转录本可以比对形成双链RNA(DsRNA)分子。我们知道dsRNA分子可以通过一种称为RNA干扰(RNAi)的降解机制来识别，这种机制会导致两种转录本的破坏。然而，我们最近发表的对正义和反义转录本分布的分析表明，没有迹象表明COPS转录本的特定降解。我们从动物系统中知道，反义转录本可以发挥比为RNAi系统形成dsRNAs更多的作用。然而，在植物中，还没有描述过这样的替代途径。因此，我们想要找出，植物有哪些替代途径，哪些需要反义转录。我们对956名警察的组织和表达的分析帮助我们做出了一个有根据的猜测，以假定反义转录的两个替代功能。(I)在COPS中，我们发现具有一个以上转录本的基因比例远远高于预期，这要么是因为从原始RNA拷贝中删除了不同的区域(替代剪接变体)，要么是因为转录本的长度不同(替代多聚腺苷基化变体)。这意味着反义转录本需要改变正义转录本的处理。(Ii)我们还经常发现，虽然正义和反义基因都被转录，但它们经常表现出对抗的行为：在许多组织类型中，一种转录型是高转录型，另一种是低转录型。对于COPS，如果特定的应激处理增加了正义转录水平，反义转录水平就会降低。然而，很快，反义转录水平增加，正义转录水平降低。对于转录水平在白天周期性变化的COPS基因(生物钟基因)，也可以看到类似的拮抗模式。这些观察表明，这是一个“转录干扰”模型，其中一个基因的转录影响另一个基因的转录，这可能被用来在诱导后重置转录水平。我们想测试反义转录本是否以及如何影响正义转录本的生产或组成。为此，我们选择单个COPS作为我们提出的两种机制中每一种的潜在模型基因。我们使用拟南芥，因为对于我们的每个COPS模型，都有一个突变系，在反义基因中插入一个。这些品系允许我们分析如果反义基因处于不活动状态，正义转录或转录本处理是否以及如何改变。我们认为，我们的工作将有助于拓宽目前对基因表达的看法，重点是转录和转录本的稳定性，但没有考虑反义转录本的调控效应。

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 the organisation 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.