PCBP2基因座位上长非编码RNA的筛选与鉴定

发布时间：2018-04-24 03:23

本文选题：ESTs + 生物信息学分析　；参考：《北京协和医学院》2013年硕士论文

【摘要】：长非编码RNA (long noncoding RNA,1ncRNA)是一类长度大于200nt,以RNA形式发挥作用的转录产物。根据1ncRNA与蛋白编码基因的相对位置,可分为：正义(sense)、反义(antisense)、双向(bidirectional)、内含子(intronic)、基因间(intergenic)五类。H Y. Chang等人近期总结了基因组范围内筛选1ncRNAs的一般策略,即通过将基因组片段上组蛋白修饰活性数据与RNA-seq数据相结合分析,寻找潜在的基因间1ncRNAs;进而对1ncRNAs的编码能力进行分析,确定它们的非编码性质。上述方法对寻找基因间1ncRNAs比较有效,但对于编码基因内部与基因同向的正义1ncRNAs和内含子1ncRNAs的筛选却存在一定的局限性。选择性剪接(Alternative Splicing)是产生蛋白质功能多样性的重要途径之一。以往研究更多关注编码基因剪接多样性,也有相关研究报道1ncRNAs也存在选择性剪接。RNA结合蛋白PCBP2在小鼠和人中均存在由选择性剪接产生的多种转录本形式,且在不同的生物学过程中发挥作用。研究报道该基因9号外显子位置有可转录1ncRNA的存在,即T-UC.338,其在肝癌中发挥重要的生物学功能,但该区域是否仅存在T-UC.338一个非编码RNA转录物尚不可知。本研究即以PCBP2内部的9号外显子区域为研究对象,探讨在该区域发现潜在1ncRNAs的生物信息学分析方法,以及该区域内1ncRNAs发生多重转录、剪接的可能。本研究从现有的数据库(UCSC http://genome.ucsc.edu/)入手,选择在同一基因座位(PCBP2的9号外显子区,UC.338基因座位)上,由内含子区域转录、剪接形成的ESTs进行鉴定和分析,并根据同源性分析结果在人和小鼠中分别进行筛选,得到四个ESTs:人源ESTs HY121526和DB276896,鼠源ESTs BQ917950和BF150035。利用Coding Potential Calculater (CPC http://cpc.cbi.pku.edu.cn)进行蛋白质编码能力分析,并将CPC分析得分与同基因座位上的对应编码基因(PCBP2)的CPC分值进行统计学比较,筛选潜在的1ncRNAs。其中HY121526、BQ917950和BF150035均显示非编码RNA的性质。同时本研究用PCR方法检测这四个ESTs以及T-UC.338在不同组织中的表达谱和在同一组织中不同时间点的表达变化来排除其为转录噪音的可能性,最终发现在人各细胞系中HY121526的表达具有一定的细胞特异性,没有检测到DB276896的表达,人源T-UC.338(H-T-UC.338)的表达也具有一定的细胞特异性；在小鼠各组织中BQ917950的表达不仅具有组织特异性,并且BQ917950在小鼠大脑发育不同时间点的表达随时间的推移呈明显下降趋势,而BF150035在小鼠各组织中广泛表达。鼠源T-UC.338(M-T-UC.338)的表达也具有组织特异性,在小鼠大脑发育不同时间点的表达随时间的推移呈现先上升后下降的趋势。本研究用RACE和步移的方法验证了H-T-UC.338的全长序列,发现H-T-UC.338的长度要超出文献报道的590bp,并检测了鼠源M-T-UC.338的全长。本研究分别做了探针,下一步将尝试用Northern的方法验证各1ncRNAs的全长。根据相关报道,1ncRNA很可能通过其所在基因座位上的编码基因起作用,并具有一定的保守性,虽然H-T-UC.338在肝癌中与PCBP2并无调控关系,但是在其他生物学事件中可能会有相关性。本研究用Real-timePCR和原位杂交实验检测了BQ917950、BF150035、 M-T-UC.338和PCBP2在小鼠发育不同时间点大脑中的表达情况,以推测基因与潜在1ncRNA之间的关系,为推测在小鼠大脑发育过程中各1ncRNAs的功能给予提示。
[Abstract]:Long non coded RNA (long noncoding RNA, 1ncRNA) is a class of transcriptional products whose length is greater than 200nt and acts in the form of RNA. According to the relative position of 1ncRNA and protein encoding genes, it can be divided into: Justice (sense), antisense (antisense), bi-directional (bidirectional), intron (intronic), and intergenic five categories The general strategy for screening 1ncRNAs in the genome scope is to search for potential INTERGENE 1ncRNAs by combining the histone modified active data on the genomic fragment with the RNA-seq data, and then analyze the encoding ability of the 1ncRNAs to determine their non coding quality. The above method is more effective for finding the INTERGENE 1ncRNAs. However, there are some limitations in the selection of 1ncRNAs and intron 1ncRNAs, which are both internally and genetically identical in coding genes.
Selective splicing (Alternative Splicing) is one of the important ways to produce protein functional diversity. Previous studies have paid more attention to the diversity of coding gene splicing, and there are also related reports that 1ncRNAs also has selective splicing.RNA binding protein PCBP2 in various transcriptional forms produced by selective splicing in mice and humans. It has been shown to play a role in different biological processes. It has been reported that the location of exon 9 of the gene has a transcriptional 1ncRNA, that is, T-UC.338, which plays an important biological function in liver cancer, but it is not known whether there is only a non coded RNA transcript of T-UC.338 in the region.
This study is based on the area of exon 9 in PCBP2, to explore the method of bioinformatics analysis of potential 1ncRNAs in this area, and the possibility of multiple transcription and splicing of 1ncRNAs in the region. This study selected the same gene seat (UCSC http:// genome.ucsc.edu/) from the existing database (UCSC http:// genome.ucsc.edu/) and selected the same gene seat (No. PCBP2). The exon region, UC.338 gene seat), was identified and analyzed by the transcriptional and splicing ESTs in the intron region, and was screened in human and mice by the results of homology analysis, and four ESTs: human sources ESTs HY121526 and DB276896 were obtained. The mouse source ESTs BQ917950 and BF150035. utilized Coding Potential Calculater. Bi.pku.edu.cn) analyzed the protein coding ability and compared the CPC analysis score with the CPC score of the corresponding coding gene (PCBP2) on the same gene seat, screening the potential 1ncRNAs. of HY121526, BQ917950 and BF150035 all showed the properties of the non coded RNA. The four ESTs and T-UC.33 were detected by PCR method at the same time. 8 the expression of expression in different tissues and changes in the expression of different time points in the same tissue exclude the possibility of transcription noise. Finally, it is found that the expression of HY121526 in human cell lines has a certain cell specificity, and the expression of DB276896 is not detected, and the expression of human T-UC.338 (H-T-UC.338) has a certain cell specific expression. The expression of BQ917950 in different tissues of mice is not only tissue specific, but the expression of BQ917950 in different time points of brain development in mice is obviously decreased with time, while BF150035 is widely expressed in various tissues of mice. The expression of mouse source T-UC.338 (M-T-UC.338) is also organized to be tissue specific in the brain of mice. The expression at different time points increased first and then decreased with time.
In this study, the full length of H-T-UC.338 was verified by RACE and step method, and the length of H-T-UC.338 was found to exceed the reported 590bp, and the length of M-T-UC.338 was detected. This study made a probe respectively. The next step will try to verify the whole length of each 1ncRNAs by Northern. According to the relevant reports, 1ncRNA is likely to pass it. The coding gene on the locus of the gene plays a role, and has a certain conservatism. Although H-T-UC.338 has no regulatory relationship with PCBP2 in liver cancer, it may be related in other biological events. This study detected BQ917950, BF150035, M-T-UC.338 and PCBP2 in mice developed by Real-timePCR and in situ hybridization. The relationship between the predicted genes and the potential 1ncRNA was discussed in order to provide a hint for the function of each 1ncRNAs in the development of mouse brain.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：Q522

【共引文献】