【作者】 唐超；

【导师】 陈路；

【作者基本信息】 四川大学 ， 遗传学， 2022， 博士

【摘要】 研究背景及思路基因表达受转录和转录后水平的调控。作为m RNA转录后的重要调控方式之一,选择性剪接(alternative splicing,AS)在细胞分化和生物体发育中发挥着重要作用。选择性剪接主要包括外显子跳跃(exon skipping,SE)、可变3’剪接位点(alternative 3’splice site,A3SS)、可变5’剪接位点(alternative 5’splice site,A5SS)以及内含子保留(intron retention,IR)等类型。其中,IR的发生导致内含子没有完成剪接而保留在成熟的m RNA中,因此内含子保留转录本(intron retention transcript,IRT)常常包含提前终止密码子(premature termination codon,PTC)而导致无义介导的降解(nonsense mediated decay,NMD),所以IR常被认为是m RNA剪接的噪音(splicing noise),而未引起足够的重视。此外,相比于其它选择性剪接事件,IR的研究还面临长度过长、序列复杂度过高以及二代测序(next generation sequencing,NGS)读长太短等诸多挑战。因此,对于IR的研究仍然存在诸多问题亟待解决。例如,IR在器官发育中起着怎样的作用,其是否具有进化上的保守性?如果IR在器官发育过程中存在保守的表达模式,它的调控机制如何?为了深入研究IR在器官发育中的功能以及调控机制,本课题利用第三代长读长测序技术对两个品系的小鼠(C57BL/6以及BALB/c)的28个器官进行了深度测序,并结合Kaessmann课题组2019年在Nature发表的包括人类、小鼠等七个物种的七个主要器官从出生前到中老年等多个时期共1,993个样本的二代测序高通量RNA-Seq数据,从进化发育生物学(evolutionary developmental biology,Evo-Devo)角度综合研究多个物种、器官、时期和技术平台中IR对于物种进化以及器官发育的功能、机制以及调控。研究结果(一)IR广泛存在于脊椎动物各器官,在器官发育中具有保守的动态变化。首先,针对小鼠多器官的三代测序数据,我们利用自主开发的IRSelector对小鼠各器官的IR进行了定量。我们发现作为m RNA转录后选择性加工事件,IR广泛存在于小鼠的28个器官中,二代测序结果进一步支持其能够影响鸟类和哺乳动物中约60-70%的基因。其次,在去除测序深度的影响后,小鼠的免疫相关的器官(thymus、c-kit~-、spleen以及c-kit~+)和神经相关的器官(cortex、hippocampus和cerebellum)中的IR水平高于其他器官,并具有较高的特异性。再次,IR具有非常高的物种以及器官特异性,能够反映物种间的进化距离、器官的发育时期,暗示IR可能参与了器官的形态发育、功能特化等过程。最后,通过神经器官与免疫器官之间的比较,我们发现器官发育过程中的转录组的调控可能由基因以及IR等转录后的动态调控共同完成。总的来说,IR作为转录后调控机制广泛参与到鸟类和哺乳动物的器官发育中,对物种进化、器官特化具有一定的贡献。(二)IR的表达水平呈现双峰分布(bimodal distribution),而低水平IR与NMD协同下调蛋白质编码基因的表达。首先,我们发现IR的表达水平呈现双峰分布(bimodal distribution),其中蛋白质编码基因的IRs的保留水平普遍偏低,而假基因或者3’UTR区域的IRs的保留水平偏高。其次,我们比较了不同保留水平的IR内含子长度、GC含量、剪接位点的强度以及编码能力,发现低水平的IRs(Low-IRs)与正常剪接的内含子更为相似,而高水平的IRs(High-IRs)则与外显子的特征具有更高相似性。因此我们推测Low-IRs与High-IRs可能存在不同的调控机制,并有以下发现支持:(a)仅有Low-IRs的保留水平与基因表达呈负相关关系;(b)在小鼠不同器官中平均有67.94%的Low-IRs导致蛋白质编码基因成为NMD的底物(PC-NMD),而High-IRs中仅有5.31%;(c)发育过程中动态变化的IRs(developmental dynamic IRs,Dev IRs)富集于Low-IRs;(d)Low-IRs显著富集于器官功能直接相关基因。因此,我们推测IR根据高低水平存在两种差异化的调控机制(1)Low-IRs具有被成功剪接的条件,其有可能在应对细胞外刺激时快速完成剪接并行使翻译蛋白质的功能;Low-IRs与NMD相耦合调控蛋白质编码基因的表达是一个保守的机制,其与发育过程中器官的功能形成与特化密切相关。(2)High-IRs在进化上受到较高的选择压并且多发生在基因的3’UTR区域,表明其可能包含基因3’UTR区域的顺式调节元件,例如mi RNA结合位点或者m6A修饰位点;此外,约1/5的High-IRs位于已知的CDS区域,表明High-IRs具有翻译成新的蛋白质的潜力。(三)外显子连接复合物(exon junction complex,EJC)核心因子参与IR的调控。为了研究IR调控中的重要基因,我们利用共表达(co-expression)分析发现在多物种的二代或三代测序数据中,EJC核心因子与Low-IRs之间存在极显著的正相关性;其中e IF4A3的CLIP信号在NMD-IRs的上游出现的频率显著高于非NMD底物的IRs;在Hep G2细胞系的敲低e IF4A3实验中,有302个显著差异的IRs事件,其中大多数(90.73%)的IRs的水平升高了。因此,e IF4A3基因表达与器官的Low-IRs比例间的相关分析、CLIP数据的富集分析以及sh RNA敲低实验均支持EJC核心因子e IF4A3与Low-IRs的调控有关。此外,我们还发现HNRNPK作为IR的调控因子,其在不同物种的不同器官中均与Low-IR的发生有关。最后,我们以大脑中P4HTM基因为例,发现其基因表达与IR呈现负相关,而该IR事件可能来源于该IR的剪接位点在啮齿类以及灵长类动物中5’剪接位点由GT到GC的变异。综上,我们从多物种、多器官、多时期、多技术平台的角度综合研究了IR对于物种进化以及器官发育的影响及其调控。我们发现Low-IR可以在转录后水平上施加一个额外的基因调控水平,以促进细胞对环境变化做出快速的反应,这其中主要包括免疫以及神经应激等;我们还发现EJC核心因子与Low-IR之间稳定的调控关系。总之,本研究为理解IR这一重要的细胞转录后调控机制在器官发育中的功能以及调控机制提供了新的线索。更多还原

【Abstract】 Research background and designGene expression is regulated at transcriptional and post-transcriptional levels.Intron retention(IR)is one of the types of alternative splicing that regulates the cell differentiation and dysregulation of this may cause the diseases.Intron retention tends to introduce premature termination codon(PTC)and is subjected to nonsense-mediated decay(NMD),therefore,IR was thought to be a result of splicing noise.However,it is largely unknown the extent and functionality of IR in the development of mammalian organs.We hypothesized that if IR is functional,it will be(a)conserved in the same organ during the evolution,(b)dynamically regulated during organ development,and(c)regulated under a fine-tuned network.To deepen our understanding of IR in mammalian organ development,here we performed the third generation full-length sequencing from Oxford Nanopore Technologies(ONT)in up to 28 organs of two strains of mice(C57BL/6 and BALB/c).Furthermore,we utilized the 1,993 RNA-seq from Next-generation sequencing(NGS)in 7 organ from 7 species from chicken to human to further test our hypothesis.Results and conclusions(A)IR is widely spread and conserved from chicken to mammalsTo better analyze IR from RNA-seq from the ONT sequencing,we developed IRselector to identify and quantify the IR from long reads.First,we found that IR is commonly used in 28 organs in mice.It is estimated that 60-70% of genes undergo IR based on short-read sequencing.Second,using the random selection to achieve the same sequencing depth,the immune and neural systems showed the highest levels of IR when compared to other organs.Finally,IR is regulated in a organ-specific manner,reflecting the evolution distance of species and the development stages in the PCA,implying that IR is a conserved regulated mechanism in organ development.(A)IR has a bimodal distribution and only low-IRs are subjected to NMDUsing the IRs from the long-read sequencing,we assessed their distribution and identified a bimodal distribution.Interestingly,this distribution showed different patterns among the organs and gene categories.The Low-IRs,that were under the 0.2in terms of IR level(IRL),tends to enrich in protein-coding genes,while the High-IRs,that were IRL greater than 0.8,are more likely to be pseudogene or be occurring in 3’UTR.Next,we compared the features including the intron length,GC content,splicing site entropy,and coding potentials between low-and High-IRs.We found that the LowIRs have similar characteristics to the introns,while the High-IRs resemble exons,indicating they may have different origins.We,therefore,hypothesized that the Lowand High-IRs may be regulated under distinct mechanisms.First,we found that the Low-IRs have a negative correlation with the host gene’s expression.Moreover,the majority of Low-IRs(67.94%)are subjected to NMD,while only a fraction(5.31%)of High-IRs are predicted to be NMD.Second,Low-IRs are more likely to be dynamically regulated during organ development and are enriched in organ-related pathways.Thus,our results suggested that IRs have two different regulatory mechanisms depending on the level of IRs.The first type is that the low-IRs are "intron-like" and could be spliced and then translated to increase protein expression when stimulated.Alternatively,these low-IRs are subjected to NMD and as a result to reduce the protein expression.The second type is that high-IRs is "exon-like" and under evolutionary constraint.Moreover,majority of them occur in the 3 ’UTR in which may contain cis-regulatory elements,such as micro RNA binding and m6 A sites,while the other 1/5 of them may potentially be translated to novel proteins.(C)Exon junction complex(EJC)core factors may be one of the important IR regulators.To identify the potential key regulators of IRs,we used the co-expression network to analyze both NGS and ONT datasets.The genes from EJC are positively correlated with low-IRs.For example,the CLIP signals from e IF4A3 are significantly enriched in the NMD-related IRs when comparing to non-NMD-related IRs.Moreover,the knock down experiment of in e IF4A3 in Hep G2 cell line indicated 302 significantly different retained IRs,and most of them(90.73%)had a higher IRL than the wild type.Together,the correlation of e IF4A3 gene expression and the proportion of Low-IRs in organs,the enriched CLIP signal,and the knockdown experiments support that the EJC core factor e IF4A3 may function as one of the regulators of Low-IRs.In addition,we also found that a splicing factor,HNRNPK,was associated with Low-IRs in organs from multiple species.Finally,we took the P4 HTM gene in the brain as an example and showed that its gene expression was negatively correlated with IR,and the IR event may be originated from the mutation of the splice site of this IR from GT to GC at the 5’ splice site from rodents.In summary,we have comprehensively investigated the effects and regulation of IR throughout the development stages of multiple organs from 7 species.We found that Low-IRs are regulated and interacted with gene regulation to promote rapid cellular responses to environmental changes,which is most evidently in immune and neural systems.We found potential regulatory relationships between EJC core factors or splicing factor and Low-IR.In conclusion,our study provides potential clues to understand the function and regulation mechanisms of IR in organ development through evolution.更多还原