Abstract：
Several MICRORNA genes belonging to same family or different families are often found in homologous or non-homologous clusters. Among the various classes, head-to-head arranged genes form one of the largest categories of non-canonically organized genes. Such head-to-head arranged, non-canonically organized genes possibly share cis-regulatory region with the intergenic sequence having the potential to function as bi-directional promoter (BDP). The transcriptional regulation of head-to-head arranged genes, especially with bidirectional promoters, remains an enigma. In the past, bidirectional promoters have been characterized for a small set of protein-coding gene pairs in plants; however, to the best of our knowledge, no such study has been carried so far for MICRORNA genes. The present study thus functionally characterizes bidirectional promoters associated with members of MIR395 family, which is evolutionary conserved and is most frequently occurring cluster across plant kingdom. In Arabidopsis thaliana, the MIR395 gene family contains six members with two head-to-head arranged gene pairs- MIR395A-B and MIR395E-F. This organization was found to be conserved at seven loci for MIR395A-B, and eleven loci for MIR395E-F in five Brassica sps. Sequence analysis of the putative bidirectional promoters revealed variation in length, GC content and distribution of strict TATA-box. Comparatively higher level of conservation at both the ends of the bidirectional promoters, corresponding to ca. 250 bp upstream of 5\'end of the respective MIRNA precursor, was observed. These conserved regions harbour several abiotic stress (nutrient, salt, drought) and hormone (ABA, ethylene) responsive cis-motifs. Functional characterization of putative bidirectional promoters associated with MIR395A-B and MIR395E-F from Arabidopsis and their respective orthologs from Brassica juncea (Bj_A08 MIR395A-B, Bj_B03 MIR395A-B, Bj_A07.1 MIR395E-F and Bj_A07.2 MIR395E-F) was carried out using a dual-reporter vector with β-glucuronidase (GUS) and Green Fluorescent Protein (GFP). Analysis of transcriptional regulation of the two reporter genes - GUS and GFP during developmental stages confirmed their bidirectional nature. Orientation-dependent differential reporter activity indicated asymmetric nature of the promoters. Comparison of the reporter activity amongst orthologs, paralogs and homeologs revealed regulatory diversification, an outcome expected in polyploid genomes. Interestingly, reporter gene activities driven by selected bidirectional promoters were also observed in anther and siliques apart vegetative tissues indicating role of miR395 in anther and fruit development. Finally, we evaluated the activity of reporter genes driven under transcriptional regulation of bidirectional promoters under normal and sulfate-deprived conditions which revealed asymmetric inducibility under sulfate-starvation, in agreement with the known role of miR395 in sulfate homeostasis.
摘要：
属于同一家族或不同家族的若干微RNA基因通常存在于同源或非同源簇中。在各种班级中，头对头排列的基因是最大的非规范组织基因之一。如此面对面的安排，非规范组织的基因可能与具有双向启动子（BDP）功能的基因间序列共享顺式调节区。头对头排列基因的转录调控，特别是双向启动子，仍然是个谜.在过去,双向启动子已被表征为植物中的一小部分蛋白质编码基因对;然而，据我们所知,到目前为止，尚未对微小RNA基因进行过这样的研究。因此，本研究在功能上表征了与MIR395家族成员相关的双向启动子，这是进化保守的，是植物界中最常见的集群。在拟南芥中，MIR395基因家族包含六个成员，具有两个头对头排列的基因对-MIR395A-B和MIR395E-F。发现该组织在MIR395A-B的七个基因座处保守，以及五个芸苔属植物中MIR395E-F的11个基因座。推定的双向启动子的序列分析揭示了长度的变化，严格的TATA盒的GC含量和分布。双向启动子两端的保守水平相对较高，对应于CA。各MIRNA前体5'末端上游250bp,被观察到。这些保守的地区有几种非生物胁迫(养分，盐,干旱)和激素(ABA，乙烯)响应性顺式基序。与拟南芥的MIR395A-B和MIR395E-F相关的推定双向启动子及其来自芥菜的直系同源物的功能表征（Bj_A08MIR395A-B，Bj_B03MIR395A-B,Bj_A07.1MIR395E-F和Bj_A07.2MIR395E-F）使用具有β-葡糖醛酸糖苷酶（GUS）和绿色荧光蛋白（GFP）的双报告载体进行。两个报告基因-GUS和GFP在发育阶段的转录调控分析证实了它们的双向性质。定向依赖性差异报告活性表明启动子的不对称性质。直系同源物之间的报告活性比较，旁系物和同源物揭示了监管多元化，多倍体基因组中预期的结果。有趣的是,在花药和角果中，除了营养组织外，还观察到了由选定的双向启动子驱动的报告基因活性，表明miR395在花药和果实发育中的作用。最后,我们评估了在正常和硫酸剥夺条件下双向启动子转录调控下驱动的报告基因的活性，这些报告基因揭示了在硫酸饥饿下的不对称诱导性，与miR395在硫酸盐稳态中的已知作用一致。
