RNA结合蛋白HLP1与HLP2调控拟南芥开花时间的分子机理研究

发布时间：2018-05-11 10:26

本文选题：开花 + RNA结合蛋白　；参考：《山东农业大学》2016年博士论文

【摘要】：开花作为植物由营养生长向生殖生长的转变,既决定了植物能否成功地繁衍后代影响自身种群的延续,同时也是一个可以影响到农作物产量和质量的重要农艺性状。植物的开花时间受到复杂而精细的调控,涉及外界环境因子,如温度和光照,以及植物自身内在因子对开花信号的识别与传递的共同作用。该过程包含了一系列基因的表达调控,从而确保植物在最合适的状态下完成这一生物学过程的转换。近期研究表明,除了四个经典的开花调控通路外,许多参与RNA转录后加工的RNA结合蛋白(RNA binding protein,RBP)或RNA加工因子参与植物开花时间的调控。RNA结合蛋白在真核生物中广泛存在,参与调控了RNA加工代谢的各个层面,包括5′-端加帽、可变剪接、RNA编辑、3′-末端多聚腺苷酸化、核质穿梭、RNA稳定性等过程,最终调控了基因的表达与生命体生长发育。拟南芥基因组编码约200个RNA结合蛋白,越来越多的研究表明,在开花时间转换、逆境响应与染色质修饰等过程中,RBP都扮演着重要的角色。前体mRNA 3′-末端的多聚腺苷酸化在真核生物基因表达与调控中具有重要的作用,该过程包含两步紧密相联的生化过程:首先在前体mRNA的多聚腺苷酸化位点(poly(A)sites,PAS)进行切割,然后添加poly(A)尾巴。Poly(A)位点的选择是多变的,该现象称之为选择性多聚腺苷酸化(Alternative polyadenylation,APA),前体mRNA通过APA产生具有不同编码序列或者是不同3′-UTR区域的转录本,增加了转录组的复杂性。大规模研究显示,APA是一种在多物种间广泛存在的基因表达调控机制,多数基因包含一个以上的多聚腺苷酸化位点,其中酵母、哺乳动物和拟南芥中高达70%以上的基因具有APA现象。APA介导的基因表达在控制机体发育、疾病发生、植物开花与环境响应等过程中具有重要的作用。APA受到了复杂而精细的调控,涉及到许多蛋白与核酸间的相互作用,然而APA在植物中的具体调控机制尚不清楚。本实验室前期在筛选植物开花相关突变体时发现At5G40490的缺失突变可以导致晚花,该基因编码一个RNA结合蛋白,进化树分析发现该蛋白与人类中的hnRNP A1和酵母中的Hrp1高度同源,将其命名为HLP1。为了解析HLP1的作用机制,采用紫外交联免疫沉淀结合大规模测序的技术在全基因组水平上鉴定HLP1所结合的靶RNA。生物信息分析显示,HLP1在全基因组水平上具有广泛的结合位点,RIP-qRT-PCR有效证明了HLP1与靶RNA结合的真实性。进一步的生物信息分析显示,HLP1结合序列显著富集在3′-UTR区域和内含子区域,并且具有两个明显的序列特征,一个是富含腺嘌呤的5′-AGAAAA-3′,另外一个是富含尿嘧啶的5′-UUUUCU-3′,它们分布于转录终止位点附近。基因功能富集分析显示,HLP1的结合基因开花过程明显相关。HLP1所结合的序列特征与poly(A)位点信号高度相似,暗示着HLP1可能参与APA的调控。poly(A)位点测序(poly(A)sites sequencing,PAS-seq)显示,HLP1的缺失突变会导致许多基因的APA转换。结合CLIP-seq分析发现,很多发生APA转换的基因是HLP1的直接靶RNA,暗示着HLP1可以直接参与APA调控。其中,HLP1可以结合在开花促进因子FCA的3′-UTR区域和第三个内含子处,促进FCA选择远端poly(A)位点,产生有功能的转录本,抑制FLC的表达,促进植物开花。序列比对发现,拟南芥中存在一个与HLP1高度同源的蛋白,二者蛋白结构域类似,均含有两个RNA识别结构域(RNA recognition motif,RRM),我们将其命名为HLP2。表型分析发现,hlp2-1突变体中FLC的表达量上升,呈现晚花表型,暗示着HLP2对植物开花时间的调控依赖于FLC。此外,我们没有鉴定到hlp1-1与hlp2-1的双突变体。进一步研究表明,hlp1hlp2双突变体胚胎致死,暗示了HLP1与HLP2在植物生长发育过程中的重要作用。亚细胞定位与核质分离实验显示HLP1与HLP2均为细胞核定位蛋白,GUS染色分析表明HLP1与HLP2表达模式一致,暗示着二者存在功能上的相关性。由于HLP1参与调控APA,以At3G15450和At3G23030为例验证,发现HLP2的缺失突变导致与HLP1缺失突变类似的poly(A)位点的转换,暗示着HLP1与HLP2均参与了对poly(A)位点的选择调控,并且二者在APA调控方面具有协同性。进一步研究发现,HLP1与HLP2均可以结合到FCA转录本的第三个内含子和3′-UTR区域,协同促进FCA转录本远端poly(A)位点的选择,促进植物开花。生化分析显示,HLP1与HLP2存在相互作用,暗示着植物体内二者以异源二聚体的形式行使功能。有意思的是,HLP1可以通过调控HLP2的APA负调控HLP2的基因表达。HLP2前体mRNA具有APA现象,正常情况下,HLP1可以结合到HLP2的前体mRNA上,抑制选择远端poly(A)位点,即抑制功能型HLP2转录本产生,负调控HLP2的基因表达。综上所述,该研究工作对拟南芥中两个未知RNA结合蛋白HLP1与HLP2的功能研究发现HLP1与HLP2可以协同作用,通过直接结合到一些RNA分子上,调控靶RNA的APA,最终调控植物开花。该工作为后续开展RBP介导的RNA加工事件在植物发育调控中的作用机制提供了新的视野。
[Abstract]:As the transformation from vegetative growth to reproductive growth, flowering determines whether plants can successfully propagate their offspring to affect the continuity of their own population, but also an important agronomic character that can affect the yield and quality of crops. The flowering time of plants is regulated by complex and fine fines, involving environmental factors, such as temperature and Light, and the interaction of the plant's own intrinsic factors to the identification and transmission of the flowering signal. This process includes a series of gene expression regulation to ensure that the plant performs this biological process in the most appropriate state. Recent studies have shown that many of the RNA transcripts are involved in addition to the four classics of the flowering regulation pathway. The post processed RNA binding protein (RNA binding protein, RBP) or RNA processing factors participate in plant flowering time regulation.RNA binding proteins widely exist in eukaryotes, participating in the regulation of the various levels of RNA processing and metabolism, including 5 '- end caps, variable splicing, RNA editing, 3' - terminal polyadenylation, nuclear shuttle, RNA stability, etc. There are about 200 RNA binding proteins encoded in Arabidopsis thaliana genome. More and more studies have shown that RBP plays an important role in the process of flowering time conversion, adversity response and chromatin modification. The polyadenylation of the precursor mRNA 3 'terminal is acidified in eukaryotic genes. It plays an important role in expression and regulation, which includes two closely linked biochemical processes: first, the polyadenylation site (poly (A) sites, PAS) of the precursor mRNA is cut, and the selection of poly (A) tail.Poly (A) loci is changeable, which is called selective polyadenylation of adenosine (Alternative polyadenylatio). N, APA), precursor mRNA produces a transcriptional transcript with different coding sequences or different 3 '-UTR regions through APA, which increases the complexity of the transcriptional group. Large-scale studies show that APA is a regulatory mechanism for gene expression widely existed among multiple species. Most genes contain more than one polyadenosine acidification site, in which yeast, lactating More than 70% of the genes in the plant and Arabidopsis have APA.APA mediated gene expression in the process of controlling body development, disease, plant flowering and environmental response..APA is regulated by complex and fine regulation, involving the interaction between many proteins and nucleic acids. However, the specific modulation of APA in plants The control mechanism is not clear. In the early stage of screening plant flowering related mutants, the deletion mutation of At5G40490 was found to lead to late flowers. The gene encodes a RNA binding protein. Phylogenetic tree analysis found that the protein was highly homologous to the Hrp1 in human hnRNP A1 and yeast, and named it HLP1. to analyze HLP1. By using the technique of UV cross-linking immunoprecipitation and large-scale sequencing, the identification of target RNA. bioinformatics combined with HLP1 showed that HLP1 had a wide binding site at the whole genome level. RIP-qRT-PCR effectively demonstrated the true reality of the combination of HLP1 and target RNA. Further bioinformatics analysis showed that HLP1 The binding sequence is significantly enriched in the 3 '-UTR region and the intron, and has two distinct sequence characteristics. One is 5' -AGAAAA-3 'of adenine rich, and the other is 5' -UUUUCU-3 'rich in uracil. They are distributed near the transcriptional terminating site. Gene function enrichment analysis shows that the HLP1 binding gene has blooming over Cheng Ming The sequence characteristics associated with the explicit correlation.HLP1 are highly similar to the poly (A) site signals, suggesting that HLP1 may participate in the regulation of.Poly (A) site sequencing of APA (poly (A) sites sequencing, PAS-seq) shows that many genes are transformed by the deletion mutation. RNA, suggesting that HLP1 can be directly involved in APA regulation, in which HLP1 can combine the 3 '-UTR region of the flowering promoting factor FCA and third introns, promote the FCA selection of the distal poly (A) site, produce a functional transcript, inhibit the expression of FLC, and promote the flowering of the plants. The protein domain is similar to the two protein domain, which all contain two RNA identification domains (RNA recognition motif, RRM). We named it to HLP2. phenotypic analysis. The expression of FLC in the hlp2-1 mutant is rising, showing the late flower phenotype, suggesting that HLP2's regulation of flowering time of the plant depends on FLC. besides FLC.. We have not identified hlp1-1 and HLP. 2-1 of the double mutants. Further study showed that hlp1hlp2 double mutant embryos died, suggesting the important role of HLP1 and HLP2 in plant growth and development. Subcellular localization and nuclear qualitative separation experiments showed that both HLP1 and HLP2 were nuclear localizing proteins, and GUS staining analysis showed that HLP1 and HLP2 expression patterns were consistent, suggesting the existence of functions of the two. As HLP1 participates in the regulation of APA, as a result of At3G15450 and At3G23030, it is found that the deletion mutation of HLP2 leads to the transformation of the poly (A) site similar to the HLP1 deletion mutation, suggesting that both HLP1 and HLP2 are involved in the selection and regulation of poly (A) sites, and the two are synergistic in the regulation. The third introns and 3 '-UTR regions of the FCA transcript can be combined with HLP2 to promote the selection of the distal poly (A) loci of the FCA transcript and promote the flowering of plants. Biochemical analysis shows that there is interaction between HLP1 and HLP2, suggesting that two of the plants in the plant exercise their function in the form of a heterogenous two polymer. The gene expression of.HLP2 precursor mRNA of HLP2 controlled by the control of HLP2 has APA phenomenon. Under normal circumstances, HLP1 can bind to the precursor mRNA of HLP2, inhibit the selection of the distal poly (A) site, that is, the inhibition of functional HLP2 transcript production and negative regulation of gene expression. In summary, the research works on two unknown binding proteins in Arabidopsis thaliana. The functional study of P1 and HLP2 found that HLP1 and HLP2 can play a synergistic role in regulating the APA of target RNA by direct binding to some RNA molecules and ultimately regulating the flowering of plants. This work provides a new field of vision for the subsequent development of RBP mediated RNA processing events in the regulation of plant development.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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