【作者】 熊慧萍；

【导师】 李飞；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2008， 硕士

【摘要】 MicroRNA（miRNA）是一类分布广泛,大小约为22个核苷酸的内源性非编码RNA,参与基因的转录后调控。部分miRNA基因分布在基因组中的基因间区,而另一些miRNA基因位于蛋白编码基因的内含子中。目前,对miRNA基因转录的了解仍十分有限,而对位内含子区域的miRNA及其与宿主基因的转录关系,更是知之甚少。有意思的是,有些miRNA基因位于宿主基因的可变剪接区域。其中,由于宿主基因的可变转录起始位点,果蝇miR-281-1/2基因位于宿主ODA基因的内含子区或5’UTR区。本论文主要研究了miR-281-1/2的转录,探讨了位于内含子区的miRNA与其宿主基因可变转录起始位点之间的关系,并进一步开展了miRNA基因的启动子分析。本项研究对更好地理解miRNA基因的转录调控,具有重要的意义。一、果蝇miR-281基因的转录及全长分析RT-PCR成功扩增出miR-281-1/2的前体,进一步利用RACE技术克隆了miR-281的全长。RACE结果表明,miR-281可能有3个转录起始位点,分别为TSS1,TSS2和TSS3。TSS1上游350bp有一个TATA盒,所有转录本具有相同的3’端,并在3’端有多核腺苷酸加尾信号（AATAAA）。实验证实,miR-281并不是从宿主基因ODA基因的内含子区加工形成,而是具有自身的独立转录本。通过与UCSC基因组数据库比对,发现三个转录起始位点的保守性均比较高。二、miR-281的二级结构,靶标预测和分子进化分析Dme-miR-281基因属于mir-46家族,包括两个前体,四个（三种）成熟体。两前体在茎区只有一个核苷酸的差异,而环区有13个核苷酸的差异。MiRBase中dme-miR-281有7个同源基因,对应于8个miRNA基因成熟体,分别属于6个物种。利用RNAfold和Mfold软件,分别对miR-281-1/2的前体和初级转录本的二级结构进行了预测,证实三个初级转录本均可形成茎环结构。利用PicTar和miRanda软件预测miR-281-1/2的靶标,发现miR-281-1有83个靶标mRNA基因,miR-281-2有69个靶标mRNA基因,miR-281-1和miR-281-2拥有共同的61个靶标基因。UCSC数据库比对结果证实,其他16个物种中存在dme-miR-281的同源序列,但miR-281前体的重复事件只发生在黑腹果蝇及其近缘物种中（果蝇科）。利用MrBayes构建分子系统进化树发现,miR-281-1和miR-281-2处于两个不同的分支。三、miR-281的表达谱及与ODA基因选择性剪接的关系利用RT-PCR的方法研究了ODA基因3个剪切变体和miR-281在不同龄期的表达谱。结果表明,3个剪切体RA、RB、RC在果蝇的每个龄期都表达,而miR-281仅在3龄幼虫,蛹和成虫期表达。荧光定量PCR结果表明,RA和RC的丰度较高,在各个龄期的变化很大。RB和miR-281的表达量较低,miR-281在不同龄期没有明显的区别。这些结果表明,ODA基因的可变转录起始位点对处于其内含子区的miR-281的转录没有明显的影响。EST分析的结果与定量PCR相对表达量的结果一致,匹配到RA和RC的EST远远多于RB和miR-281。四、miR-281基因的启动子及Myc转录因子的免疫共沉淀分析利用TRANSFAC数据库预测发现,TSS1,TSS2和TSS3附近有重要的转录因子结合位点如:Myc,C/EBP,CAD等等。TSS1上下游有两个Myc结合位点。通过染色质免疫共沉淀实验,证明Myc结合于miR-281基因启动子区,可能参与miR-281的转录调节,这也间接证明了miR-281的独立转录。UCSC数据库比对结果显示,两个Myc转录因子结合位点的保守性较高。将TSS1上游1020 bp及下游412 bp的区域作为可能的启动子区,构建了pMiR-281-GFP载体,转染果蝇S2细胞,并利用CuSO₄促进转录表达,结果未能发现明显的绿色荧光。分析可能存在多个原因,一是miR-281启动子的启动效率太低,以致观察不到GFP发出的荧光;二是果蝇S2细胞不表达miR-281;三是选择构建载体的启动子区域不是真正的启动子区。五、多重转录起始位点的转录效率分析利用随机引物进行反转录的cDNA为模板,进行定量PCR来分析miR-281基因不同转录起始位点的转录效率。在3龄幼虫期,与转录本1比较,转录本2是转录本1的0.97倍,在成虫期,转录本2是转录本1的0.83倍。这一结果表明,TSS1和TSS2具有相似的转录效率。转录本3在3龄幼虫期和成虫期分别是转录本1的4.34倍和1.33倍,表明TSS3的转录效率比TSS1和TSS2都高,但TSS3随着果蝇的不同发育阶段,转录效率有所变化。更多还原

【Abstract】 MicroRNA(miRNA) is an abundant class of small(～22 nucleotides),endogenous noncoding RNAs that function at post-transcriptional gene regulation.Some miRNAs are located in the intergenic region,whereas other miRNAs are intronic.Until now,little has been known about the transcription of intronic miRNA and its relationship with host gene. We found that some intronic miRNAs are located in the alternative regions of host genes. For example,Drosophila miR-281 cluster is in either the first intron or the 5’UTR of the ODA gene.Here,the transcription of miR-281 and its relationship with the host gene were studied.The promoter of miR-281 was also analyzed.This work is helpful for better understanding the complex and fine-tuning regulation of miRNA transcription.1.Drosophila miR-281 transcription analysisThe precursor of miR-28 lcluster was successfully detected by RT-PCR.The full length of the miR-281 transcripts was amplified with the RACE technique.According to RACE results,the transcripts of miR-281 were produced by three different transcription start sites, named as TSS1,TSS2 and TSS3.A TATA box is found at the 350 bp upstream of TSS1.All detected transcripts were polyadenylated,with a canonical polyadenylation signal (AATAAA) near 3’ terminus.The conservation of genomic regions flanking three TSSs are higher than average levels.Though miR-281 is located in the intron of ODA gene,it is independently transcribed with its own promoter.2.Secondary structure analysis,target predicted and evolution analysis of miR-281Dme-miR-281 gene that includes two precursors and four mature miRNA belongs to mir-46 gene family.Only one nucleotide variance exists at the arm regions of pre-miR-281-1 and pre-miR-281-2,whereas up to 13 nucleotide changes appear in the loop region.In the miRBase,there are seven homolog precurors(corresponding to eight homolog mature miRNA) of dme-miR-281.RNAFold and Mfold were used to predict the secondary structure of pre-miR-281 and pri-miR-281.In the structures of all three transcripts,pre-miRNA regions fold into typical hairpin structures.Eight-three protein coding genes were predicted to be the target of miR-281-1,and 69 mRNAs were targeted by miR-281-2.Among two groups of mRNA targets,61 mRNAs were targeted by both miR-281-1 and miR-281-2.The homologs of miR-281 are found in sixteen species from the UCSC database. Interestingly,the duplication of pre-miR-281 only appears in Drosophila melanogaster and its close species.In the miR-46 family tree inferred by the MrBayes algorithm,miR-281-1 and miR-281-2 are clustered into two different clades.3.Expression profile analysis and the relationship with its host geneExpression analysis showed that isoform RA,RB and RC of ODA gene expressed at every stage,whereas the transcripts of pri-miRNA281 were only detected in the third instar, pupa and adult.Real-time PCR showed that the abundance of isoform RA and RC is higher than that of isoform RB and miR-281.This is consistent with EST analysis results.There are more ESTs supporting isoforms RA and RC than RB and miR-281.The expression of isoform RA and RC varied at different development stages.However,there was no significant difference of the miR-281 among the different stages.These results suggested that alternative transcription start sites of the host ODA genes have little impact on the transcription of intronic miR-281 cluster.4.The promoter analysis of miR-281 and ChIP analysis of MycThe genomic reigons -1020 bp to +412 bp flanking the TSS were defined as putative promoter.By searching the TRANSFAC database,some well-known transcription factors such as Myc,C/EBP and CAD are predicted to bind to putative the promoter region of dme-miR-281.The binding sites are highly conserved between diverse organisms by Blaring the UCSC database.The genomic DNA binding with Myc protein was immunicipated by Chromatin Immunoprecipitation(CHIP) technique.ChlP-PCR with miR-281 primers confirmed the physical presence of Myc at the promoter region of miR-281.This suggested that Myc might participate in the transcription of miR-281 and provided a piece of evidence of independently transcription of intronic miR-281.The putative promoter ranging from -1020 bp and +412 bp flanking the TSS1 were amplified and ligated with pAC5.1 vector that contains green Fluorescent protein(GFP) as the report gene.The constructs were transected into the Drosophila S2.However,the fluorence of GFP protein could not be detected,indicating that the putative miR-281 promoter did not drive the expression of downstream GFE One possible reason is that miR-281 promoter has a low activity in Drosophila S2 cell as miR-281 only expresses at the third instar,pupa and adult stage.5.Efficiency analysis of multiple transcription start sites of miR-281To estimate the transcriptional efficiency of different TSSs,the real-time PCRs were carried out using isoform-specific primers.Two sets of cDNA template were produced.One is reversed transcribed using random hexamers as anchor primer.Another is reverse transcribed with Oligo(dT) primer.The results manifested that TSS3 might be a genuine TSS.Relative to TSS1 transcripts,TSS2 transcripts is 0.97 fold in the third instar and 0.83 fold at adult,suggesting that TSS1and TSS2 have similar transcriptional efficiency.The transcriptional efficiency of TSS3 is changable at different development stages.Generally, it has a higher efficiency than TSS1 and TSS3.更多还原