,1, 赵欣1, 张策1, 刘威,2Fragile X mental retardation protein participates in non-coding RNA pathways
Enhui Li,1, Xin Zhao1, Ce Zhang1, Wei Liu,2编委: 夏昆
收稿日期:2017-11-7修回日期:2018-01-5网络出版日期:2018-01-09
| 基金资助: |
Received:2017-11-7Revised:2018-01-5Online:2018-01-09
| Fund supported: |
作者简介 About authors
作者简介:李恩惠,硕士研究生,专业方向：神经生理学E-mail:
通讯作者:刘威,博士,副教授,研究方向：发育生物学E-mail:
摘要
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李恩惠, 赵欣, 张策, 刘威. 脆性X智力低下蛋白参与非编码RNA通路的研究进展. 遗传[J], 2018, 40(2): 87-94 doi:10.16288/j.yczz.17-255
Enhui Li, Xin Zhao, Ce Zhang, Wei Liu.
脆性X综合征(Fragile X syndrome)是一种最常见的遗传性智力低下疾病,患者最明显的特征是中、重度智力障碍。其发病率在男性中约为1/4000,在女性中约为1/8000,并且在不同种族中均有发病病例[1]。脆性X综合征是由脆性X智力低下基因(Fragile X mental retardation 1, FMR1)突变而导致其编码的脆性X智力低下蛋白(Fragile X mental retardation protein, FMRP)缺失或者功能受损造成的[2]。在人体发育过程中,FMRP在不同的组织中广泛表达,其中以脑和生殖腺中表达量最多,因此FMRP突变能显著影响大脑和睾丸的生理活动[3]。FMRP是一类功能上高度保守的蛋白质,dFMRP是其在果蝇(Drosophila)体内唯一的同系物。最新研究发现,在果蝇中FMRP能够通过非编码RNA(non-coding RNA)通路而发挥广泛的功能[4,5],其功能和价值正引起人们的关注。
非编码RNA是生物体内重要的基因表达调控分子,影响着许多生理活动,并影响着许多疾病的发生和发展。目前研究发现非编码RNA主要有4大类：siRNA(small interfering RNA)、miRNA(microRNA)、piRNA(Piwi-interacting RNA)和lncRNA(long non- coding RNA)。这些非编码RNA在体内通过参与不同的信号通路,可从转录、转录后及表观遗传等水平上调控基因表达,从而在体内发挥着重要调控作用[6]。本文综述了FMRP参与4种非编码RNA通路,重点讨论了与piRNA相互作用,以期促进人们从新的角度认识脆性X综合征,加深对脆性X综合征发病机制的认识。
1 脆性X综合征与FMRP
多名****几乎同时发现脆性X综合征是由位于Xq27.3位点的单基因FMR1基因突变所致[7,8,9]。FMR1编码的蛋白质FMRP在许多组织中表达,特别在神经和生殖系统中表达更丰富。除此之外,哺乳动物FMRP还有两个同源蛋白FXR1P和FXR2P (>60%氨基酸同源性)[10]。相比FMRP,FXR1P表达分布更加广泛,在大脑、小脑、睾丸、骨骼肌和心肌等组织中均有表达,并且在骨骼肌和心脏发育中发挥重要作用,其突变会导致遗传性肌肉萎缩症[11,12]。FXR2P在体内也广泛表达,并与FMRP在神经发育、突触活动和代谢活动等过程中发挥协同作用[13]。黑腹果蝇(Drosophila melanogaster)是生命科学研究的经典模式生物,常用于揭示不同生物学过程的分子细胞机制。dFmr1是FMR1在果蝇体内的同源基因,而且是果蝇基因组编码的唯一同源基因,在结构上与人FMR1高度保守[14]。dFmr1突变体也出现神经系统与生殖系统等缺陷,这与脆性X综合征病人对应的临床病症类似,说明FMRP在功能上具有高度保守性。综上所述,果蝇是研究脆性X综合征的有效模型,可以帮助人们揭示其致病的分子机理。FMRP是一个著名的RNA结合蛋白,含有3个高度保守的RNA结合结构域：2个KH结构域和1个RGG盒(图1)[15]。最初研究发现,KH结构域特异识别mRNA环-环“假结”(loop-loop pseudoknot)三级结构,因此KH突变将降低其与环-环“假结”mRNA亲和力[16]。同样,RGG盒能够结合mRNA G-四连体(G-quartet)结构[17]。除了RNA结合域,FMRP还有1个核定位信号(NLS)和1个核输出信号(NES)。借助NLS进入神经元细胞核,FMRP通过KH结构域和RGG盒与特定RNA转录子组装成信使核糖核蛋白(mRNP)复合物,借助NES进入细胞质,进而在细胞质中调节靶基因的翻译[18]。FMRP通过与mRNA结合,在Dicer酶和Argonaute(Ago)家族蛋白等协同下,可以调控体内约70% mRNA翻译,因此FMRP是一个在转录后水平上调控基因表达的重要蛋白质[19]。
图1
新窗口打开|下载原图ZIP|生成PPT图1人与果蝇脆性X智力低下蛋白的结构比较
人与果蝇FMRP总体同源性和一致性,蛋白各个结构域相对应的一致性[14]。NLS：核定位信号;KH1：KH结构域1;KH2：KH结构域2;NES：核输出信号;RGG：精氨酸甘氨酸簇。
Fig. 1Structural comparison of human and Drosophila FMRP
2 FMRP与siRNA和miRNA
FMRP参与多个非编码RNA通路,最先发现的为siRNA和miRNA通路[20]。siRNA是20~25个核苷酸的双链小分子RNA,依赖于RNA诱导沉默复合物(RNA-induced silencing complex,RISC)而参与转录后调控。在此过程中Dicer酶和Ago2蛋白被认为是RNA干扰的标志性结构。Ishizuka等[21]首先在果蝇中发现了FMRP和(Ago2蛋白、RNA解旋酶Dmp68、Dicer酶)存在于同一复合体中,表明FMRP参与siRNA通路之间存在相互作用。Caudy等[22]在生物化学水平证明了FMRP与RISC组分免疫共沉淀。进一步研究发现FMRP是RISC的组成成分之一,然而其在RISC中的功能尚未表明。综上所述,FMRP与RISC相关组分结合,参与了siRNA通路。miRNA是20~25个核苷酸的单链小分子RNA。果蝇体内,dFMRP能够与Dicer酶和Ago1等互作,调节miRNA的加工和成熟,进而调控靶基因的表达。dFmr1基因敲除会导致神经特异的miR-124a表达水平下降[23]。随着研究的深入,发现越来越多的miRNA与FMRP关系密切,包括神经系统中miRNA-1和miRNA-281[24]。这些研究表明,FMRP通过miRNA通路在神经元发育过程中发挥至关重要作用。在果蝇卵巢内,dFMRP可与bantam miRNA互作来调控生殖干细胞的命运[25]。综上所述,FMRP可参与miRNA通路,在机体神经活动、生殖干细胞的命运决定等进程中发挥着重要作用。
3 FMRP与piRNA
3.1 piRNA生物合成与功能
piRNA是与Piwi蛋白相作用的RNA,长度约为23~32个核苷酸。piRNAs主要来源于含有大量转座子和重复序列的基因间区(intergenic region),又称之为piRNA簇[26],目前研究发现piRNA簇主要有单向转录和双向转录两种类型。在果蝇中,从其基因组的起源和与Ago蛋白的结合研究中,能够洞察piRNA的合成机制。piRNA合成路径可分为初级通路和次级通路(图2)。初级通路中,piRNA簇转录产物进入胞浆,经核酸酶Zuc剪切与加工形成成熟piRNA,并在Yb小体上结合Piwi蛋白,形成piRNA- Piwi复合物[27]。次级通路中,初级piRNA经过Aub与Ago3等加工,piRNA得到大量扩增,生成次级piRNA [28]。生成的piRNA能够通过碱基互补方式与转座子序列反义链结合,在转录水平上沉默转座子,并进一步维持基因组稳定[29]。在此过程中,piRNA发挥作用需要许多协助蛋白,例如Aub、Ago3、Vasa和Tudor等。重要的是,piRNA通路在果蝇和哺乳动物的生殖细胞中具有高度保守性,相关蛋白突变体内转座子和重复序列表达比野生型更活跃,常常降低了生育能力[30,31]。3.2 FMRP参与piRNA通路
已有研究发现,FMRP和piRNA通路相关蛋白可以形成复合体,例如成人睾丸中的FMRP与piRNA通路的一个关键分子Vasa共定位,也与piNG体(piRNA nuage giant body)共定位[33,34]。上面的研究提供了一些间接证据,提示FMRP可能参与piRNA通路。2015年,Bozzetti等[5]首先报道dFMRP与piRNA通路的关键蛋白Aub和Ago1相互作用。dFMRP通过Tudor结构域识别Aub和Ago1,并与之N末端相结合[5]。这些结果首次证实了dFMRP参与piRNA通路。随后,本研究团队发现,dFMRP与piRNA通路的另一个关键蛋白Piwi互作,而且这种互作不依赖于RNA[35]。dFMRP通过Tudor结构域识别Piwi,并与之N末端相结合。体外实验结果显示,dFMRP的N端比C端与Piwi结合力更强,并且只与Piwi N末端相结合。此外,dFMRP在piwi突变体卵巢内表达量显著降低,表明dMFRP的表达依赖Piwi,提示dFMRP可能在Piwi下游发挥作用。综上所述,dFMRP与piRNA通路相关蛋白Aub、Ago1和Piwi等相互作用,参与piRNA通路。图2
新窗口打开|下载原图ZIP|生成PPT图2piRNA合成途径
在体细胞中,单向转录的转录产物进入胞浆,经核酸酶Zuc剪切与加工形成成熟piRNA,并在Yb小体上结合Piwi蛋白形成piRNA-Piwi复合物[27]。在生殖细胞中,存在piRNA初级通路和次级通路两种通路。初级通路中,单向和双向转录的piRNA簇转录产物进入胞浆,与体细胞内类似,经过加工形成初级piRNA,完成初级通路。初级piRNA通过DEAD盒(DEAD-box)解旋酶UAP56被运输至piNG体进一步加工[32]。在次级通路中,初级piRNA经过Aub、Ago3的加工,使piRNA得到大量扩增,生成次级piRNA[28]。Zuc：核酸酶;Piwi：Piwi蛋白;Aub：Aubergine蛋白;Ago3：Argonaute-3蛋白;Hsp83：果蝇体内Hsp90蛋白的同源物;Vasa：DEAD盒RNA解旋酶;Hen1：甲基转移酶。
Fig. 2The biosynthetic pathway of piRNA
3.3 FMRP调控转座子表达
piRNA通过碱基互补与转座子序列反义链结合,可在转录水平上沉默转座子。Bozzetti等[5]发现roo, I, R1, ZAM等转座子在dFmr1Δ50和dFmr1Δ113雄性突变体睾丸中表达量增加,但Su(Ste)piRNA的表达量大幅减少。同样,本研究团队发现mdg1, roo, HeT-A, I-element, mst40等逆转录转座子和重复序列在dFmr1突变体卵中表达增加(表1),而roo piRNA表达水平却降低,表明dFMRP和Piwi协同沉默转座子和重复序列[35]。综述所述,dFMRP和Aub、Piwi等piRNA通路相关蛋白互作,调控转座子和重复序列的表达。3.4 FMRP维持染色质结构和功能
piRNA是维护染色质正常结构和功能所必需的。piRNA通过碱基互补与靶序列结合,招募染色质修饰相关蛋白,如HP1和组蛋白甲基转移酶Su(var)3- 9。这些复合体在转座子及其基因组周围建立抑制性组蛋白标记,如H3K9Me3等,以维护异染色质基因沉默[41]。的确,本研究团队发现HP1在dFmr1突变体卵巢异染色质中定位出现异常,提示FMRP在染色质维护中发挥作用。在野生型中,HP1聚集于着丝粒染色质上,且呈规则的半月状,而HP1在dFmr1突变果蝇卵泡细胞呈散点分布,说明dFMRP参与异染色质的维护[42]。结合piRNA功能,这些研究结果提示dFMRP与Piwi、Aub、Ago1等蛋白结合,通过直接piRNA-DNA互补序列,介导识别特定靶序列,然后招募表观遗传因子,在转座子及其基因组周围建立抑制性组蛋白标记,以维护异染色质基因沉默[41](图3)。可以推测,dFMRP通过piRNA通路,抑制转座子及重复序列活性,从而维持细胞内基因组稳定,保证果蝇生育能力。的确,Piwi; dFmr1双突变体中卵巢发育缺陷和原始生殖细胞的异常分化更严重,进一步证实了上述观点[35]。Table 1
表1
表1 dFmr1突变体中与piRNA通路相关的转座子表达异常
Table 1
| 转座子 | 主要分布 | 功能 | 参考文献 |
|---|---|---|---|
| mdg1 | 常染色质,Y染色体 | 干扰、提前终止转录;形成反义转录本 | [36] |
| roo | 常染色质 | 调控真核生物生殖系基因组结构 | [37] |
| HeT-A | 线性染色体端粒 | 延长端粒;果蝇生殖系内piRNA的靶点 | [38] |
| I-element | 常染色质 | 参与反义piRNA的形成 | [39] |
| mst40 | 2号染色体左臂接近异染色质区 | 形成反义转录本 | [40] |
| R1 | 生殖细胞特异 | 参与piRNA的形成 | [5] |
| ZAM | 体细胞特异 | 参与piRNA的形成 | [5] |
新窗口打开|下载CSV
4 FMRP与lncRNA
lncRNA是一类长度大于200个核苷酸的非编码RNA。越来越多的研究表明FMRP与lncRNA有着密切关系。Pastori等[43]发现,FMR1启动子上游序列转录生成正义链lncRNA FMR5,而FMR6是包含FMR1 3°UTR区的反义链lncRNA,而且二者在脆性X综合征患者和基因前突变携带者中表达水平有差异。因此,FMR5与FMR6可能是新的分子标志物,可用于脆性X综合征的筛查、诊断和治疗。最新研究发现,lncRNA TUG1的表达在FMR1基因敲除小鼠大脑中显著升高[44]。RNA-免疫共沉淀实验结果显示,FMRP能够与TUG1直接结合,并降低其稳定性,从而负向调控TUG1的表达。高表达TUG1导致神经元的轴突长度下降,影响神经元树突及树突棘发育和突触可塑性,表明FMRP可以通过TUG1而特异性调控神经元轴突发育。这为从lncRNA水平了解、干预脆性X综合征提供借鉴与参考。图3
新窗口打开|下载原图ZIP|生成PPT图3FMRP通过参与piRNA通路维护异染色质结构和功能
dFMRP与Piwi结合,通过piRNA与DNA序列互补,介导dFMRP与Piwi蛋白识别特定靶序列,并且招募表观遗传因子HP1和Su(var)3-9,修饰DNA和组蛋白,以维护异染色质基因沉默。FMRP：脆性X智力低下蛋白;Piwi：Piwi蛋白;Su(var)3-9：组蛋白甲基转移酶;HP1：异染色质蛋白;Me：甲基化;H3K9：组蛋白甲基化;Pol-Ⅱ：聚合酶Ⅱ。
Fig. 3FMRP maintains the structure and function of heterochromatin by piRNA pathways
近年来关于lncRNA调控基因表达的具体分子机制逐步被破解,也为从lncRNA通路了解脆性X综合征的发病机制提供了新思路。在表观遗传水平, lncRNA可调控组蛋白的甲基化和乙酰化等修饰,也可以影响DNA启动子区CpG岛的甲基化水平和染色质重构等,进而调控基因的表达[45]。而脆性X综合征与FMR1 5°端CpG岛异常甲基化有关[46],那么lncRNA是否通过调控FMR1 CpG岛的甲基化水平而调控FMR1转录有待进一步研究。此外,lncRNA作为非编码小分子RNA的前体,可被Dicer酶或Drosha酶剪切成miRNA和piRNA等,进而间接调控基因的表达[47]。本研究团队已发现FMRP通过piRNA通路沉默转座子及重复序列的表达[35]。lncRNA作为piRNA的前体,FMRP是否与相关蛋白协同参与lncRNA加工,从而影响lncRNA来源的piRNA生物合成?这一推测尚有待进一步验证。
5 结语和展望
脆性X综合征作为一种智力低下疾病,从首次发现致病基因FMR1开始,研究人员从不同角度揭示了该病的分子病理,逐步形成一个复杂的网络,对于脆性X综合征的发病机制和临床应用具有重要意义。机体生命活动都依赖于DNA、RNA及蛋白之间形成错综复杂的调控网络,因此未来的研究将趋向于从整体和系统的角度去研究生物体的生命过程。科研人员利用动物模型,对FMRP参与非编码RNA通路进行深入研究,并揭示了FMRP在神经系统活动、生殖干细胞命运决定和染色质稳定性等方面具有重要作用。由于FMRP的结构与功能及非编码RNA通路在进化上均具有高度保守性,因此推测在脆性X综合征中也有类似重要的调节作用。这些研究从非编码RNA水平上为脆性X综合征的发病机理提供了新见解,或许成为临床应用一个新的突破口,为预防和治疗该疾病带来了新希望。目前,亟需开展临床医学研究,形成交叉研究,进一步验证非编码RNA在脆性X综合征发病机制中作用。尽管目前对FMRP全部功能和发病详细机制仍然不清楚,也尚无有效的预防和治疗方法,但人类对其研究和探索不会止步,通过科研工作者不懈努力,相信终有一天会破解其中的奥秘。参考文献 原文顺序
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被引期刊影响因子
URLPMID:5557344 [本文引用: 1]
Fragile X syndrome (FXS) is an autistic intellectual disability disorder caused by loss of the RNA binding protein FMRP. Treating FXS has been challenging because of the lack of a reliable in vivo drug screening model. Kashima et al . found that the hyperactive locomotion observed in a fly model of FXS was a reliable behavioral marker for the neurological abnormalities underlying the disease. The kinase LIMK1 is implicated in the pathogenesis of FXS, and high-throughput (rapid, quantitative, and time- and cost-effective) drug screening in the fly FXS model confirmed that LIMK1 inhibitors ameliorated both the neurological and behavioral phenotypes of this model. LIMK1 inhibitors also reduced hyperactivity in a mouse model of FXS. Thus, this method may aid in future drug development for FXS patients.
URLPMID:4216185 [本文引用: 1]
Fragile X Syndrome (FXS) is considered the most common form of inherited intellectual disability. It is caused by reductions in the expression level or function of a single protein, the Fragile X Mental Retardation Protein (FMRP), a translational regulator which binds to approximately 4% of brain messenger RNAs. Accumulating evidence suggests that FXS is a complex disorder of cognition, involving interactions between genetic and environmental influences, leading to difficulties in acquiring key life skills including motor skills, language, and proper social behaviors. Since many FXS patients also present with one or more features of autism spectrum disorders (ASDs), insights gained from studying the monogenic basis of FXS could pave the way to a greater understanding of underlying features of multigenic ASDs. Here we present an overview of the FXS and FMRP field with the goal of demonstrating how loss of a single protein involved in translational control affects multiple stages of brain development and leads to debilitating consequences on human cognition. We also focus on studies which have rescued or improved FXS symptoms in mice using genetic or therapeutic approaches to reduce protein expression. We end with a brief description of how deficits in translational control are implicated in FXS and certain cases of ASDs, with many recent studies demonstrating that ASDs are likely caused by increases or decreases in the levels of certain key synaptic proteins. The study of FXS and its underlying single genetic cause offers an invaluable opportunity to study how a single gene influences brain development and behavior.
[本文引用: 1]
URLPMID:22843500 [本文引用: 1]
Fragile X syndrome, the most common form of inherited mental retardation, is caused by the loss of the fragile X mental retardation protein (FMRP). FMRP is a ubiquitously expressed, multi-domain RNA-binding protein, but its in vivo function remains poorly understood. Recent studies have shown that FMRP participates in cell cycle control during development. Here, we used Drosophila mutants to test if FMRP plays a role in DNA damage response under genotoxic stress. We found significantly fewer dfmr1 mutants survived to adulthood than wild-types following irradiation or exposure to chemical mutagens, demonstrating that the loss of drosophila FMRP (dFMRP) results in hypersensitivity to genotoxic stress. Genotoxic stress significantly reduced mitotic cells in wild-type brains, indicating the activation of a DNA damage-induced G2/M checkpoint, while mitosis was only moderately suppressed in dfmr1 mutants. Elevated expression of cyclin B, a protein critical for the G2 to M transition, was observed in the larval brains of dfmr1 mutants. CycB mRNA transcripts were enriched in the dFMRP-containing complex, suggesting that dFMRP regulates DNA damage-induced G2/M checkpoint by repressing CycB mRNA translation. Reducing CycB dose by half in dfmr1 mutants rescued the defective G2/M checkpoint and reversed hypersensitivity to genotoxic stress. In addition, dfmr1 mutants exhibited more DNA breaks and elevated p53-dependent apoptosis following irradiation. Moreover, a loss-of-heterozygosity assay showed decreased irradiation-induced genome stability in dfmr1 mutants. Thus, dFMRP maintains genome stability under genotoxic stress and regulates the G2/M DNA damage checkpoint by suppressing CycB expression.
URLPMID:25908854 [本文引用: 6]
RNA metabolism controls multiple biological processes, and a specific class of small RNAs, called piRNAs, act as genome guardians by silencing the expression of transposons and repetitive sequences in the gonads. Defects in the piRNA pathway affect genome integrity and fertility. The possible implications in physiopathological mechanisms of human diseases have made the piRNA pathway the object of intense investigation, and recent work suggests that there is a role for this pathway in somatic processes including synaptic plasticity. The RNA-binding fragile X mental retardation protein (FMRP, also known as FMR1) controls translation and its loss triggers the most frequent syndromic form of mental retardation as well as gonadal defects in humans. Here, we demonstrate for the first time that germline, as well as somatic expression, of Drosophila Fmr1 (denoted dFmr1), the Drosophila ortholog of FMRP, are necessary in a pathway mediated by piRNAs. Moreover, dFmr1 interacts genetically and biochemically with Aubergine, an Argonaute protein and a key player in this pathway. Our data provide novel perspectives for understanding the phenotypes observed in Fragile X patients and support the view that piRNAs might be at work in the nervous system.
URLMagsci [本文引用: 1]
表观遗传学是研究基因表达发生了可遗传的改变, 而DNA序列不发生改变的一门生物学分支, 对细胞的生长分化及肿瘤的发生发展至关重要。表观遗传学的主要机制包括DNA甲基化、组蛋白修饰及新近发现的非编码RNA。非编码RNA 是指不能翻译为蛋白的功能性RNA分子, 其中常见的具调控作用的非编码RNA包括小干涉RNA、miRNA、piRNA 以及长链非编码RNA。近年来大量研究表明非编码RNA在表观遗传学的调控中扮演了越来越重要的角色。文章综述了近年来生物细胞非编码RNA调控的表观遗传学研究进展, 以有助于理解哺乳动物细胞中非编码RNA及其调控机制和功能。
URLMagsci [本文引用: 1]
表观遗传学是研究基因表达发生了可遗传的改变, 而DNA序列不发生改变的一门生物学分支, 对细胞的生长分化及肿瘤的发生发展至关重要。表观遗传学的主要机制包括DNA甲基化、组蛋白修饰及新近发现的非编码RNA。非编码RNA 是指不能翻译为蛋白的功能性RNA分子, 其中常见的具调控作用的非编码RNA包括小干涉RNA、miRNA、piRNA 以及长链非编码RNA。近年来大量研究表明非编码RNA在表观遗传学的调控中扮演了越来越重要的角色。文章综述了近年来生物细胞非编码RNA调控的表观遗传学研究进展, 以有助于理解哺乳动物细胞中非编码RNA及其调控机制和功能。
URLPMID:1710175 [本文引用: 1]
Fragile X syndrome is the most frequent form of inherited mental retardation and is associated with a fragile site at Xq27.3. We identified human YAC clones that span fragile X site-induced translocation breakpoints coincident with the fragile X site. A gene (FMR-1) was identified within a four cosmid contig of YAC DNA that expresses a 4.8 kb message in human brain. Within a 7.4 kb EcoRI genomic fragment, containing FMR-1 exonic sequences distal to a CpG island previously shown to be hypermethylated in fragile X patients, is a fragile X site-induced breakpoint cluster region that exhibits length variation in fragile X chromosomes. This fragment contains a lengthy CGG repeat that is 250 bp distal of the CpG island and maps within a FMR-1 exon. Localization of the brain-expressed FMR-1 gene to this EcoRI fragment suggests the involvement of this gene in the phenotypic expression of the fragile X syndrome.
URLPMID:8515814 [本文引用: 1]
The fragile X syndrome is the most frequent form of inherited mental retardation after Down's syndrome, having an incidence of one in 1,250 males. The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene. This CGG repeat shows length variation in normal individuals and is increased significantly in both carriers and patients; it is located 250 base pairs distal to a CpG island which is hypermethylated in fragile X patients. The methylation probably results in downregulation of FMR-1 gene expression. No information can be deduced about the function of the FMR-1 protein from its predicted sequence. Here we investigate the nature and function of the protein encoded by the FMR-1 gene using polyclonal antibodies raised against the predicted amino-acid sequences. Four different protein products, possibly resulting from alternative splicing, have been identified by immunoblotting in lymphoblastoid cell lines of healthy individuals. All these proteins were missing in cell lines from patients not expressing FMR-1 messenger RNA. The intracellular localization of the FMR-1 gene products was investigated by transient expression in COS-1 cells and found to be cytoplasmic. Localization was also predominantly cytoplasmic in the epithelium of the oesophagus, but in some cells was obviously nuclear.
URL [本文引用: 1]
Fragile X mental retardation syndrome is caused by the unstable expansion of a CGG repeat in the FMR-1 gene. In patients with a full mutation, abnormal methylation results in suppression of FMR-1 transcription. FMR-1 is expressed in many tissues but its function is unknown. We have raised monoclonal antibodies specific for the FMR-1 protein. They detect 4-5 protein bands which appear identical in cells of normal males and of males carrying a premutation, but are absent in affected males with a full mutation. Immunohistochemistry shows a cytoplasmic localization of FMR-1. The highest levels were observed in neurons, while glial cells contain very low levels. In epithelial tissues, levels of FMR-1 were higher in dividing layers. In adult testis, FMR-1 was detected only in spermatogonia. FMR-1 was not detected in dermis and cardiac muscle except under pathological conditions.
URLPMID:20442204 [本文引用: 1]
Fragile X syndrome (FXS), resulting solely from the loss of function of the human fragile X mental retardation 1 (hFMR1) gene, is the most common heritable cause of mental retardation and autism disorders, with syndromic defects also in non-neuronal tissues. In addition, the human genome encodes two closely related hFMR1 paralogs: hFXR1 and hFXR2. The Drosophila genome, by contrast, encodes a single dFMR1 gene with close sequence homology to all three human genes. Drosophila that lack the dFMR1 gene (dfmr1 null mutants) recapitulate FXS-associated molecular, cellular and behavioral phenotypes, suggesting that FMR1 function has been conserved, albeit with specific functions possibly sub-served by the expanded human gene family. To test evolutionary conservation, we used tissue-targeted transgenic expression of all three human genes in the Drosophila disease model to investigate function at (1) molecular, (2) neuronal and (3) non-neuronal levels. In neurons, dfmr1 null mutants exhibit elevated protein levels that alter the central brain and neuromuscular junction (NMJ) synaptic architecture, including an increase in synapse area, branching and bouton numbers. Importantly, hFMR1 can, comparably to dFMR1, fully rescue both the molecular and cellular defects in neurons, whereas hFXR1 and hFXR2 provide absolutely no rescue. For non-neuronal requirements, we assayed male fecundity and testes function. dfmr1 null mutants are effectively sterile owing to disruption of the 9+2 microtubule organization in the sperm tail. Importantly, all three human genes fully and equally rescue mutant fecundity and spermatogenesis defects. These results indicate that FMR1 gene function is evolutionarily conserved in neural mechanisms and cannot be compensated by either FXR1 or FXR2, but that all three proteins can substitute for each other in non-neuronal requirements. We conclude that FMR1 has a neural-specific function that is distinct from its paralogs, and that the unique FMR1 function is responsible for regulating neuronal protein expression and synaptic connectivity.
URL [本文引用: 1]
BACKGROUND: The Fragile X Mental retardation-Related 1 (FXR1) gene belongs to the fragile X related family, that also includes the Fragile X Mental Retardation (FMR1) gene involved in fragile X syndrome, the most common form of inherited mental retardation. While the absence of FMRP impairs cognitive functions, inactivation of FXR1 has been reported to have drastic effects in mouse and xenopus myogenesis. Seven alternatively spliced FXR1 mRNA variants have been identified, three of them being muscle specific. Interestingly, they encode FXR1P isoforms displaying selective RNA binding properties. METHODS AND RESULTS: Since facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy characterised by altered splicing of mRNAs encoding muscle specific proteins, we have studied the splicing pattern of FXR1 mRNA in myoblasts and myotubes of FSHD patients. We show here that FSHD myoblasts display an abnormal pattern of expression of FXR1P isoforms. Moreover, we provide evidence that this altered pattern of expression is due to a specific reduced stability of muscle specific FXR1 mRNA variants, leading to a reduced expression of FXR1P muscle specific isoforms. CONCLUSION: Our data suggest that the molecular basis of FSHD not only involves splicing alterations, as previously proposed, but may also involve a deregulation of mRNA stability. In addition, since FXR1P is an RNA binding protein likely to regulate the metabolism of muscle specific mRNAs during myogenesis, its altered expression in FSHD myoblasts may contribute to the physiopathology of this disease.
URL [本文引用: 1]
Background Changes in criteria and differences in populations studied and methodology have produced a wide range of prevalence estimates for mild cognitive impairment (MCI). Methods Uniform criteria were applied to harmonized data from 11 studies from USA, Europe, Asia and Australia, and MCI prevalence estimates determined using three separate definitions of cognitive impairment. Results The published range of MCI prevalence estimates was 5.0%–36.7%. This was reduced with all cognitive impairment definitions: performance in the bottom 6.681% (3.2%–10.8%); Clinical Dementia Rating of 0.5 (1.8%–14.9%); Mini-Mental State Examination score of 24–27 (2.1%–20.7%). Prevalences using the first definition were 5.9% overall, and increased with age (P < .001) but were unaffected by sex or the main races/ethnicities investigated (Whites and Chinese). Not completing high school increased the likelihood of MCI (P ≤ .01). Conclusion Applying uniform criteria to harmonized data greatly reduced the variation in MCI prevalence internationally.
URLPMID:25552647 [本文引用: 1]
Fragile X syndrome results from loss of FMR1 expression. Individuals with the disorder exhibit not only intellectual disability, but also an array of physical and behavioral abnormalities, including sleep difficulties. Studies in mice demonstrated that Fmr1, along with its paralog Fxr2, regulate circadian behavior, and that their absence disrupts expression and cycling of essential clock mRNAs in the liver. Recent reports have identified circadian genes to be essential for normal metabolism. Here we describe the metabolic defects that arise in mice mutated for both Fmr1 and Fxr2. These mice have reduced fat deposits compared with age- and weight-matched controls. Several metabolic markers show either low levels in plasma or abnormal circadian cycling (or both). Insulin levels are consistently low regardless of light exposure and feeding conditions, and the animals are extremely sensitive to injected insulin. Glucose production from introduced pyruvate and glucagon is impaired and the mice quickly clear injected glucose. These mice also have higher food intake and higher VO2 and VCO2 levels. We analyzed liver expression of genes involved in glucose homeostasis and found several that are expressed differentially in the mutant mice. These results point to the involvement of Fmr1 and Fxr2 in maintaining the normal metabolic state in mice.
URLPMID:11733059 [本文引用: 2]
Abstract Fragile X mental retardation gene (FMR1) encodes an RNA binding protein that acts as a negative translational regulator. We have developed a Drosophila fragile X syndrome model using loss-of-function mutants and overexpression of the FMR1 homolog (dfxr). dfxr nulls display enlarged synaptic terminals, whereas neuronal overexpression results in fewer and larger synaptic boutons. Synaptic structural defects are accompanied by altered neurotransmission, with synapse type-specific regulation in central and peripheral synapses. These phenotypes mimic those observed in mutants of microtubule-associated Futsch. Immunoprecipitation of dFXR shows association with futsch mRNA, and Western analyses demonstrate that dFXR inversely regulates Futsch expression. dfxr futsch double mutants restore normal synaptic structure and function. We propose that dFXR acts as a translational repressor of Futsch to regulate microtubule-dependent synaptic growth and function.
URLPMID:28257890 [本文引用: 1]
Abstract Fragile X mental retardation protein (FMRP),an important RNA-binding protein responsible for fragile X syndrome, is involved in posttranscriptional control of gene expression that links with brain development and synaptic functions. Here, we reveal a novel role of FMRP in pre-mRNA alternative splicing, a general event of posttranscriptional regulation. Using co-immunoprecipitation and immunofluorescence assays, we identified that FMRP interacts with an alternative-splicing-associated protein RNA-binding protein 14 (RBM14) in a RNA-dependent fashion, and the two proteins partially colocalize in the nuclei of hippocampal neurons. We show that the relative skipping/inclusion ratio of the micro-exon L in the Protrudin gene and exon 10 in the Tau gene decreased in the hippocampus of Fmr1 knockout (KO) mice. Knockdown of either FMRP or RBM14 alters the relative skipping/inclusion ratio of Protrudin and Tau in cultured Neuro-2a cells, similar to that in the Fmr1 KO mice. Furthermore, overexpression of FMRP leads to an opposite pattern of the splicing, which can be offset by RBM14 knockdown. RNA immunoprecipitation assays indicate that FMRP promotes RBM14's binding to the mRNA targets. In addition, overexpression of the long form of Protrudin or the short form of Tau promotes protrusion growth of the retinoic acid-treated, neuronal-differentiated Neuro-2a cells. Together, these data suggest a novel function of FMRP in the regulation of pre-mRNA alternative splicing through RBM14 that may be associated with normal brain function and FMRP-related neurological disorders.
URLPMID:23235829 [本文引用: 1]
Abstract Fragile X syndrome (FXS) is a multi-organ disease that leads to mental retardation, macro-orchidism in males and premature ovarian insufficiency in female carriers. FXS is also a prominent monogenic disease associated with autism spectrum disorders (ASDs). FXS is typically caused by the loss of fragile X mental retardation 1 (FMR1) expression, which codes for the RNA-binding protein FMRP. Here we report the discovery of distinct RNA-recognition elements that correspond to the two independent RNA-binding domains of FMRP, in addition to the binding sites within the messenger RNA targets for wild-type and I304N mutant FMRP isoforms and the FMRP paralogues FXR1P and FXR2P (also known as FXR1 and FXR2). RNA-recognition-element frequency, ratio and distribution determine target mRNA association with FMRP. Among highly enriched targets, we identify many genes involved in ASD and show that FMRP affects their protein levels in human cell culture, mouse ovaries and human brain. Notably, we discovered that these targets are also dysregulated in Fmr1(-/-) mouse ovaries showing signs of premature follicular overdevelopment. These results indicate that FMRP targets share signalling pathways across different cellular contexts. As the importance of signalling pathways in both FXS and ASD is becoming increasingly apparent, our results provide a ranked list of genes as basis for the pursuit of new therapeutic targets for these neurological disorders.
URLPMID:11719188 [本文引用: 1]
Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.
URLPMID:8842725 [本文引用: 1]
Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggests that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP.
URLPMID:2767401 [本文引用: 1]
Abstract Noncoding RNAs play important and diverse regulatory roles throughout the genome and make major contributions to disease pathogenesis. The FMR1 gene is involved in three different syndromes: fragile X syndrome (FXS), primary ovarian insufficiency (POI), and fragile X-associated tremor/ataxia syndrome (FXTAS) in older patients. Noncoding RNAs have been implicated in the molecular pathogenesis of both FXS and FXTAS. Here we will review our current knowledge on the role(s) of noncoding RNAs in FXS and FXTAS, particularly the role of the microRNA pathway in FXS and the role of noncoding riboCGG (rCGG) repeat in FXTAS.
URLPMID:15516998 [本文引用: 1]
Fragile X syndrome is caused by the loss of an RNA-binding protein called FMRP (for fragile X mental retardation protein). FMRP seems to influence synaptic plasticity through its role in mRNA transport and translational regulation. Recent advances include the identification of mRNA ligands, FMRP-mediated mRNA transport and the neuronal consequence of FMRP deficiency. FMRP was also recently linked to the microRNA pathway. These advances provide mechanistic insight into this disorder, and into learning and memory in general.
URLPMID:12368261 [本文引用: 1]
Abstract Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.
URLPMID:12368260 [本文引用: 1]
Abstract RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. Here, we report the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). We identify two putative RNA-binding proteins, the Drosophila homolog of the fragile X mental retardation protein (FMRP), dFXR, and VIG (Vasa intronic gene), through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.
URLPMID:19005053 [本文引用: 1]
Abstract Fragile X syndrome is the most common form of inherited mental retardation caused by loss of the fragile X mental retardation protein 1 (FMRP). The detailed molecular pathways underlying the pathogenesis of this disorder remain incompletely understood. Here, we show that miR-124a, a nervous-system-specific miRNA, is associated with the Drosophila homolog of FMRP (dFMR1) in vivo. Ectopic expression of wild-type but not mutant miR-124a precursors decreased dendritic branching of dendritic arborization sensory neurons, which was partially rescued by the loss of dFMR1 activity, suggesting that the biogenesis and/or function of miR-124a are partially dependent on dFMR1. Indeed, in contrast with the complete loss of mature miR-124a in Dicer-1 mutants, steady-state levels of endogenous or ectopically expressed mature miR-124a were partially reduced in dfmr1 mutants, whereas the level of pre-miR-124a increased. This effect could be explained in part by the reduced abundance of the Dicer-1-Ago1 complex in the absence of dFMR1. These findings suggest a modulatory role for dFMR1 to maintain proper levels of miRNAs during neuronal development.
URLPMID:14703574 [本文引用: 1]
Abstract Fragile X syndrome is caused by a loss of expression of the fragile X mental retardation protein (FMRP). FMRP is a selective RNA-binding protein which forms a messenger ribonucleoprotein (mRNP) complex that associates with polyribosomes. Recently, mRNA ligands associated with FMRP have been identified. However, the mechanism by which FMRP regulates the translation of its mRNA ligands remains unclear. MicroRNAs are small noncoding RNAs involved in translational control. Here we show that in vivo mammalian FMRP interacts with microRNAs and the components of the microRNA pathways including Dicer and the mammalian ortholog of Argonaute 1 (AGO1). Using two different Drosophila melanogaster models, we show that AGO1 is critical for FMRP function in neural development and synaptogenesis. Our results suggest that FMRP may regulate neuronal translation via microRNAs and links microRNAs with human disease.
URLPMID:2654963 [本文引用: 1]
Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP). We have previously demonstrated that dFmr1, the Drosophila ortholog of the fragile X mental retardation 1 gene, plays a role in the proper maintenance of germline stem cells in Drosophila ovary; however, the molecular mechanism behind this remains elusive. In this study, we used an immunoprecipitation assay to reveal that specific microRNAs (miRNAs), particularly the bantam miRNA (bantam), are physically associated with dFmrp in ovary. We show that, like dFmr1, bantam is not only required for repressing primordial germ cell differentiation, it also functions as an extrinsic factor for germline stem cell maintenance. Furthermore, we find that bantam genetically interacts with dFmr1 to regulate the fate of germline stem cells. Collectively, our results support the notion that the FMRP-mediated translation pathway functions through specific miRNAs to control stem cell regulation.
URLPMID:25895683 [本文引用: 1]
PIWI-interacting RNAs (piRNAs) are a large family of small, single-stranded, non-coding RNAs present throughout the animal kingdom. They form complexes with several members of the PIWI clade of Argonaute proteins and carry out regulatory functions. Their best established biological role is the inhibition of transposon mobilization, which they enforce both at the transcriptional level, through regulation of heterochromatin formation, and by promoting transcript degradation. In this capacity, piRNAs and PIWI proteins are at the heart of the germline cells’ efforts to preserve genome integrity. Additional regulatory roles of piRNAs and PIWI proteins in gene expression are becoming increasingly apparent. PIWI proteins and piRNAs are often detected in human cancers deriving from germline cells as well as somatic tissues. Their detection in cancer correlates with poorer clinical outcomes, suggesting that they play a functional role in the biology of cancer. Nonetheless, the currently available information, while highly suggestive, is still not sufficient to entirely discriminate between a ‘passenger’ role for the ectopic expression of piRNAs and PIWI proteins in cancer from a ‘driver’ role in the pathogenesis of these diseases. In this article, we review some of the key available evidence for the role of piRNAs and PIWI in human cancer and discuss ways in which our understanding of their functions may be improved.
URL [本文引用: 2]
PIWI-interacting RNAs (piRNAs) protect genome integrity from transposons. In Drosophila ovarian somas, primary piRNAs are produced and loaded onto Piwi. Here, we describe roles for the cytoplasmic Yb body components Armitage and Yb in somatic primary piRNA biogenesis. Armitage binds to Piwi and is required for localizing Piwi into Yb bodies. Without Armitage or Yb, Piwi is freed from the piRNAs and does not enter the nucleus. Thus, piRNA loading is required for Piwi nuclear entry. We propose that a functional Piwi-piRNA complex is formed and inspected in Yb bodies before its nuclear entry to exert transposon silencing.
URLPMID:3236501 [本文引用: 2]
Abstract piRNAs guide PIWI proteins to silence transposons in animal germ cells. Reciprocal cycles of piRNA-directed RNA cleavage--catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila melanogaster--expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. We show that qin, a piRNA pathway gene whose protein product contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, a perinuclear structure implicated in transposon silencing. In qin mutants, less Ago3 binds Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, and DNA damage accumulates in nurse cells and oocytes. We propose that Qin enforces heterotypic Ping-Pong between Aub and Ago3, ensuring that transposons are silenced and maintaining the integrity of the germline genome.
URL [本文引用: 1]
In Drosophila gonads, Piwi proteins and associated piRNAs collaborate with additional factors to form a small RNA-based immune system that silences mobile elements. Here, we analyzed nine Drosophila piRNA pathway mutants for their impacts on both small RNA populations and the subcellular localization patterns of Piwi proteins. We find that distinct piRNA pathways with differing components function in ovarian germ and somatic cells. In the soma, Piwi acts singularly with the conserved flamenco piRNA cluster to enforce silencing of retroviral elements that may propagate by infecting neighboring germ cells. In the germline, silencing programs encoded within piRNA clusters are optimized via a slicer-dependent amplification loop to suppress a broad spectrum of elements. The classes of transposons targeted by germline and somatic piRNA clusters, though not the precise elements, are conserved among Drosophilids, demonstrating that the architecture of piRNA clusters has coevolved with the transposons that they are tasked to control.
URLPMID:17446352 [本文引用: 1]
Nearly half of the mammalian genome is composed of repeated sequences. In Drosophila, Piwi proteins exert control over transposons. However, mammalian Piwi proteins, MIWI and MILI, partner with Piwi-interacting RNAs (piRNAs) that are depleted of repeat sequences, which raises questions about a role for mammalian Piwi's in transposon control. A search for murine small RNAs that might program Piwi proteins for transposon suppression revealed developmentally regulated piRNA loci, some of which resemble transposon master control loci of Drosophila. We also find evidence of an adaptive amplification loop in which MILI catalyzes the formation of piRNA 5' ends. Mili mutants derepress LINE-1 (L1) and intracisternal A particle and lose DNA methylation of L1 elements, demonstrating an evolutionarily conserved role for PIWI proteins in transposon suppression.
URL [本文引用: 1]
Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element (ME) activity in animal germline. To date, nearly all piRNA studies have been conducted in model organisms and little is known about piRNA diversity, target specificity and biological function in human.Here we performed high-throughput sequencing of piRNAs from three human adult testis samples. We found that more than 81% of the ~17 million putative piRNAs mapped to ~6,000 piRNA-producing genomic clusters using a relaxed definition of clusters. A set of human protein-coding genes produces a relatively large amount of putative piRNAs from their 3'UTRs, and are significantly enriched for certain biological processes, suggestive of non-random sampling by the piRNA biogenesis machinery. Up to 16% of putative piRNAs mapped to a few hundred annotated long non-coding RNA (lncRNA) genes, suggesting that some lncRNA genes can act as piRNA precursors. Among major ME families, young families of LTR and endogenous retroviruses have a greater association with putative piRNAs than other MEs. In addition, piRNAs preferentially mapped to specific regions in the consensus sequences of several ME (sub)families and some piRNA mapping peaks showed patterns consistent with the "ping-pong" cycle of piRNA targeting and amplification.Overall our data provide a comprehensive analysis and improved annotation of human piRNAs in adult human testes and shed new light into the relationship of piRNAs with protein-coding genes, lncRNAs, and mobile genetic elements in human.
URLPMID:23141543 [本文引用: 1]
Abstract piRNAs silence transposons during germline development. In Drosophila, transcripts from heterochromatic clusters are processed into primary piRNAs in the perinuclear nuage. The nuclear DEAD box protein UAP56 has been previously implicated in mRNA splicing and export, whereas the DEAD box protein Vasa has an established role in piRNA production and localizes to nuage with the piRNA binding PIWI proteins Ago3 and Aub. We show that UAP56 colocalizes with the cluster-associated HP1 variant Rhino, that nuage granules containing Vasa localize directly across the nuclear envelope from cluster foci containing UAP56 and Rhino, and that cluster transcripts immunoprecipitate with both Vasa and UAP56. Significantly, a charge-substitution mutation that alters a conserved surface residue in UAP56 disrupts colocalization with Rhino, germline piRNA production, transposon silencing, and perinuclear localization of Vasa. We therefore propose that UAP56 and Vasa function in a piRNA-processing compartment that spans the nuclear envelope.
URLPMID:23587717 [本文引用: 1]
Abstract DEAD-box helicases related to the Drosophila protein Vasa (also known as Ddx4) are found throughout the animal kingdom. They have been linked to numerous processes in gametogenesis, germ cell specification, and stem cell biology, and alterations in Vasa expression are associated with malignancy of tumor cells and with some human male infertility syndromes. Experimental results indicating how Vasa contributes to all these different cellular and developmental processes are discussed, using examples from planarians, Caenorhabditis elegans, Drosophila, sea urchin, zebrafish, Xenopus, mouse, and human. Molecular, cellular, and developmental functions of Vasa and its orthologs are reviewed in this article. Evidence linking Vasa to translational regulation, to biogenesis of small RNAs, and to chromosome condensation is examined. Finally, potential overlapping functions between Vasa and related DEAD-box helicases (Belle, or Ddx3, and DEADSouth, or Ddx25) are explored. This article is part of a Special Issue entitled: The biology of RNA helicases - Modulation for life.
[本文引用: 1]
URLPMID:26842990 [本文引用: 4]
Fragile X syndrome(Fra X), the most common form of inherited mental retardation, is caused by the absence of the evolutionally conserved fragile X mental retardation protein(FMRP). While neuronal functions of FMRP have been intensively studied for the last two decades, its role in non-neuronal cells remains poorly understood. Piwi, a key component of the Piwi-interacting RNA(pi RNA) pathway,plays an essential role in germline development. In the present study, we report that similar to piwi, dfmr1, the Drosophila homolog of human FMR1, is required for transposon suppression in the germlines. Genetic analyses showed that dfmr1 and piwi act synergistically in heterochromatic silencing, and in inhibiting the differentiation of primordial germline cells and transposon expression. Northern analyses showed that roo pi RNA expression levels are reduced in dfmr1 mutant ovaries, suggesting a role of dfmr1 in pi RNA biogenesis.Biochemical analysis demonstrated a physical interaction between d FMRP and Piwi via their N-termini. Taken together, we propose that d FMRP cooperates with Piwi in maintaining genome integrity by regulating heterochromatic silencing in somatic cells and suppressing transposon activity via the pi RNA pathway in germlines.
URLPMID:3074071 [本文引用: 1]
The mdg1 retrovirus-like retrotransposon of Drosophila melanogaster was found to possess a complex promoter which can be transcribed by both RNA polymerases II and III (pol II and pol III). Pol III transcription, which is not typical of protein-coding genes, is driven by the sequences located in the long terminal repeat (LTR) of mdg1, predominantly within the transcribed region and is initiated 10 bp upstream from the regular pol II RNA start site. The pol III RNA start site is observed not only in in vitro transcription reactions, but also in total RNA isolated from tissue culture cells, larvae, pupae and adult flies. A possible role of pol III transcription in mechanisms controlling the expression of full-length mdg1-encoded transcripts in the developing fly, which are apparently relaxed in cell culture, is discussed.
[本文引用: 1]
URLPMID:20 [本文引用: 1]
Background Telomere replication in Drosophila depends on the transposition of a domesticated retroelement, the itHeT-A /itretrotransposon. The sequence of the itHeT-A /itretrotransposon changes rapidly resulting in differentiated subfamilies. This pattern of sequence change contrasts with the essential function with which the itHeT-A /itis entrusted and brings about questions concerning the extent of sequence variability, the telomere contribution of different subfamilies, and whether wild type and mutant Drosophila stocks show different itHeT-A /itscenarios. Results A detailed study on the variability of itHeT-A /itreveals that both the level of variability and the number of subfamilies are higher than previously reported. Comparisons between GIII, a strain with longer telomeres, and its parental strain Oregon-R indicate that both strains have the same set of itHeT-A /itsubfamilies. Finally, the presence of a highly conserved splicing pattern only in its antisense transcripts indicates a putative regulatory, functional or structural role for the itHeT-A /itRNA. Interestingly, our results also suggest that most itHeT-A /itcopies are actively expressed regardless of which telomere and where in the telomere they are located. Conclusions Our study demonstrates how the itHeT-A /itsequence changes much faster than previously reported resulting in at least nine different subfamilies most of which could actively contribute to telomere extension in Drosophila. Interestingly, the only significant difference observed between Oregon-R and GIII resides in the nature and proportion of the antisense transcripts, suggesting a possible mechanism that would in part explain the longer telomeres of the GIII stock.
URL [本文引用: 1]
Germline silencing of transposable elements is essential for the maintenance of genome integrity. Recent results indicate that this repression is largely achieved through a RNA silencing pathway that involves Piwi-interacting RNAs (piRNAs). However the repressive mechanisms are not well understood. To address this question, we used the possibility to disrupt the repression of the Drosophila I element retrotransposon by hybrid dysgenesis. We show here that the repression of the functional I elements that are located in euchromatin requires proteins of the piRNA pathway, and that the amount of ovarian I element piRNAs correlates with the strength of the repression in the female germline. Antisense RNAs, which are likely used to produce antisense piRNAs, are transcribed by heterochromatic defective I elements, but efficient production of these antisense small RNAs requires the presence in the genome of euchromatic functional I elements. Finally, we demonstrate that the piRNA-induced silencing of the functional I elements is at least partially posttranscriptional. In a repressive background, these elements are still transcribed, but some of their sense transcripts are kept in nurse cell nuclear foci together with those of the Doc retrotransposon. In the absence of I element piRNAs, either in dysgenic females or in mutants of the piRNA silencing pathway, sense I element transcripts are transported toward the oocyte where retrotransposition occurs. Our results indicate that piRNAs are involved in a posttranscriptional gene-silencing mechanism resulting in RNA nuclear accumulation.
URLPMID:17381314 [本文引用: 1]
Abstract We adapted the nuclear run-on method to measure changes in the rate of RNA polymerase II (pol II) transcription of repetitive elements and transposons in the female germ line of Drosophila melanogaster. Our data indicate that as little as an approximately 1.5-fold change in the rate of transcription can be detected by this method. Our nuclear run-on protocol likely measures changes in transcriptional elongation, because rates of transcription decline with time, consistent with a low rate of pol II re-initiation in the isolated nuclei. Surprisingly, we find that the retrotransposon gypsy and the repetitive sequence mst40 are silenced posttranscriptionally in fly ovaries.
URLPMID:3504300 [本文引用: 2]
Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression.
URLPMID:18245345 [本文引用: 1]
The sticky/citron kinase protein is a conserved regulator of cell-cycle progression from invertebrates to humans. While this kinase is essential for completion of cytokinesis, sticky/citron kinase phenotypes disrupting neurogenesis and cell differentiation suggest additional non-cell-cycle functions. However; it is not. known whether these phenotypes are an indirect consequence of sticky mutant cell-cycle defects or whether they define a novel function for this kinase. We have isolated a temperature-sensitive allele of the Drosophila sticky gene and we show that sticky/citron kinase is required for histone H3-K9 methylation, HPI localization, and heterobit-omatin-mediated gene silencing. sticky genetically interacts with Argonaute 7 and sticky mutants exhibit context-dependent St(var) and E(var) activity. These observations indicate that sticky/citron kinase functions to regulate both actin-myosin-mediated cytokinesis and epigenetic gene silencing, possibly linking cell-cycle progression to heterochromatin assembly and inheritance of gene expression states.
URL [本文引用: 1]
The majority of the human genome is transcribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, >200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains <55 CGG repeats in its 5'-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called "Deep-RACE", which combines rapid amplification of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 promoter, whereas FMR6 is an antisense transcript overlapping the 3' region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clinical outcomes.
URLPMID:29211876 [本文引用: 1]
Abstract LncRNAs have recently emerged to influence the pathogenesis of fragile X syndrome (FXS), which is caused by the functional loss of fragile X mental retardation protein (FMRP). However, the interaction between FMRP and lncRNAs on regulating neuronal development remains elusive. Here, we reported that FMRP directly interacted with lncRNA TUG1, and decreased its stability. Furthermore, TUG1 bond to transcriptional regulator, SnoN, and negatively modulated SnoN-Ccd1 pathway to specifically control axonal development. These observations suggested interplay between FMRP and lncRNAs might contribute to the pathogenesis of FXS.
URL [本文引用: 1]
长链非编码RNA(long non-coding RNA, lncRNA)是一类转录本长度超过200nt、不编码蛋白质的RNA。近年来,随着染色质构象捕获及转录组测序等技术的发展,lncRNA与染色质构象间的关系越来越受到重视。多项研究表明,lncRNA在基因调控网络中具有重要的作用,可通过影响细胞核高级结构的动态变化来调控真核基因的表达。因其广泛的基因调控功能及在肿瘤发生过程中的重要作用,lncRNA被认为是未来肿瘤临床诊断和预后判定的新型标志物之一。本文旨在介绍lncRNA改变细胞核高级结构从而调控关键基因表达的分子机制,并详细介绍lncRNA在肿瘤治疗中的临床意义。
URL [本文引用: 1]
长链非编码RNA(long non-coding RNA, lncRNA)是一类转录本长度超过200nt、不编码蛋白质的RNA。近年来,随着染色质构象捕获及转录组测序等技术的发展,lncRNA与染色质构象间的关系越来越受到重视。多项研究表明,lncRNA在基因调控网络中具有重要的作用,可通过影响细胞核高级结构的动态变化来调控真核基因的表达。因其广泛的基因调控功能及在肿瘤发生过程中的重要作用,lncRNA被认为是未来肿瘤临床诊断和预后判定的新型标志物之一。本文旨在介绍lncRNA改变细胞核高级结构从而调控关键基因表达的分子机制,并详细介绍lncRNA在肿瘤治疗中的临床意义。
URLPMID:27585064 [本文引用: 1]
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. It is most frequently caused by an abnormal expansion of the CGG trinucleotide repeat (>200 repeats) located in the promoter of the fragile X mental retardation gene ( FMR1 ), resulting in promoter DNA hypermethylation and gene silencing. Current clinical tests for FXS are technically challenging and labor intensive, and may involve use of hazardous chemicals or radioisotopes. We clinically validated the Illumina Infinium HumanMethylation450 DNA methylation array for FXS screening. We assessed genome-wide and FMR1 -specific DNA methylation in 32 males previously diagnosed with FXS, including nine with mosaicism, as well as five females with full mutation, and premutation carrier males ( n =11) and females ( n =11), who were compared to 300 normal control DNA samples. Our findings demonstrate 100% sensitivity and specificity for detection of FXS in male patients, as well as the ability to differentiate patients with mosaic methylation defects. Full mutation and premutation carrier females did not show FMR1 methylation changes. We have clinically validated this genome-wide DNAmethylation assay as a cost- and labor-effective alternative for sensitive and specific screening for FXS, while ruling out the most common differential diagnoses of FXS, Prader-Willi syndrome, and Sotos syndrome in the same assay.
URLPMID:21843590 [本文引用: 1]
Abstract Posttranscriptional gene regulation by small RNAs and its crucial impact on development, apoptosis, stem cell self-renewal and differentiation gained tremendous scientific attention since the discovery of RNA interference (RNAi) and microRNAs (miRNAs). However, in the last few years, many more examples for regulatory small RNAs were discovered, some of them even with miRNA-like functions. Even though these small RNA molecules were previously thought to be mere artifacts accumulating during the preparation of RNA libraries, advances in sequencing technology revealed that small RNAs derive from hairpin-fold RNA structures, for example. Mirtrons, short hairpin RNAs or small RNAs that are processed from longer non-coding RNAs such as tRNAs or snoRNAs have been found recently and some of them might be involved in the regulation of gene expression in different organisms. Furthermore, small RNAs originating from transposable elements, heterochromatic regions or convergent transcription units forming endogenous short interfering RNAs (endo-siRNAs) are the somatic equivalents of the germline-specific Piwi-interacting RNAs (piRNAs) in mediating transposon silencing. This review will focus on several recent findings that have added new aspects to small RNA-guided gene silencing.
