CN115216483B - poly(A)尾巴非A修饰在促进mRNA翻译中的应用 - Google Patents
poly(A)尾巴非A修饰在促进mRNA翻译中的应用 Download PDFInfo
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Abstract
本发明公开了poly(A)尾巴非A修饰在促进mRNA翻译中的应用。本发明通过分析人、小鼠细胞的poly(A)尾巴，以及mRNA的翻译，发现poly(A)尾巴非A修饰与翻译效率呈正相关。遗传学分析显示，线虫中的一种非典型poly(A)聚合酶GLD‑4可通过建立poly(A)尾巴非A修饰来调控翻译。并进一步通过实验证明poly(A)尾巴非A修饰可以有效地促进mRNA的翻译，poly(A)尾巴中的G、C、U的添加可以促进mRNA的翻译并提高相应蛋白质的含量。从而提出poly(A)尾巴非A修饰可以作为有效促进mRNA翻译的全新技术手段。
Description
技术领域
本发明涉及生物技术领域中，poly(A)尾巴非A修饰在促进mRNA翻译中的应用。
背景技术
Poly(A)尾巴影响RNA的稳定性、出核和翻译效率，因而对于RNA的代谢至关重要。然而，由于二代测序分析平台读取核酸同聚物序列的固有缺陷，大多数的转录组分析均丢失了 poly(A)尾巴信息。因此，poly(A)尾巴对于RNA的命运和功能调控是亟待解析的。研究人员使用TAIL-seq和PAL-seq(poly(A)-tail length profiling by sequencing)等方法在 Illumina平台估算了poly(A)尾巴的长度(Chang et al.,2014；Subtelny et al.,2014)。不同基因的翻译效率大不相同(Ingolia et al.,2009)。果蝇、非洲爪蟾和小鼠等多个物种的卵细胞RNA poly(A)尾巴研究显示，poly(A)尾巴长度和mRNA翻译效率之间具有明显的相关性(Eichhorn et al.,2016；Lim et al.,2016；Luong et al.,2020；Subtelny etal., 2014；Yang et al.,2020)。卵细胞中较长的poly(A)尾巴可以导致更为有效的mRNA翻译，但体细胞中却不存在相同的调控机制(Luong et al.,2020；Park et al.,2016；Subtelny et al.,2014)。出人意料的是，即使很短的poly(A)尾巴(最短20nt)也可以支持体细胞里 mRNA的翻译(Park et al.,2016)。同时也有研究观点认为体细胞中poly(A)尾巴长度只要超过20nt即可，更长的poly(A)尾巴也不会有额外的mRNA翻译调控功能(Park etal.,2016)。
TAIL-seq分析结果显示，RNA的3’末端可通过末端延伸机制掺入其他核苷酸，这种修饰目前认为可调控RNA的稳定性，从而成为了mRNA稳定性调控的另一层次(Chang etal., 2014；Chang et al.,2018；Lim et al.,2014；Lim et al.,2018；Morgan et al.,2019；Morgan et al.,2017)。去腺苷酸化的短poly(A)尾巴可被TUT4/7加上末端U修饰，这种修饰可将靶RNA分子标记并降解(Lim et al.,2014)。相反，TENT4A/B引入的G修饰可以防止 poly(A)尾巴被CCR4-NOT复合体去腺苷酸化，从而提高mRNA的稳定性(Lim et al.,2018)。
Poly(A)尾巴内部的核苷酸组成曾是一个巨大的未知，近期基于三代测序平台的PAIso-seq和FLAM-seq方法被开发出来，实现了对于poly(A)尾巴这类长核酸同聚物的读取(Legnini et al.,2019；Liu et al.,2019)。这两种方法均检测到了一种在小鼠卵子、线虫和人细胞系的poly(A)尾巴内部广泛分布的新RNA修饰：非A修饰(poly(A)Tail Internalnon-A Modifications，pATIM)(Legnini et al.,2019；Liu et al.,2019)，然而其功能未知。不同基因含有pATIM的转录本比例也大不相同，这也引出了一个重要的科学问题：pATIM有什么样的功能(Arango et al.,2018；Wang et al.,2015)。pATIM功能的研究具有重要的意义。
发明内容
本发明所要解决的技术问题是如何提高mRNA翻译效率。
为解决上述技术问题，本发明首先提供了下述任一应用：
1、通过在poly(A)尾巴中掺入非A修饰在mRNA翻译或提高mRNA的翻译效率中的应用；
2、促进poly(A)尾巴非A修饰的物质在提高mRNA翻译效率中的应用；
3、促进poly(A)尾巴非A修饰的物质在制备提高mRNA翻译效率产品中的应用。
在poly(A)尾巴中掺入非A修饰可通过直接化学合成掺入非A修饰完成，也可通过生物化学方法完成，也可通过在mRNA末端连接含有非A修饰的poly(A)尾巴完成，也可通过将包括含有非A修饰的poly(A)尾巴模板的mRNA转录模板进行转录完成。
进一步，在poly(A)尾巴中掺入非A修饰可通过生物化学方法在细胞外完成，也可向细胞中导入具有促进poly(A)尾巴非A修饰功能蛋白质的编码基因完成，也即，可在细胞外完成，也可在细胞内完成。
所述促进poly(A)尾巴非A修饰的物质可为通过化学方法在poly(A)尾巴中掺入非A修饰的物质，也可为可在不含poly(A)尾巴的RNA末端连接含有非A修饰的poly(A)尾巴的物质。
所述通过生物化学方法在poly(A)尾巴中掺入非A修饰的物质可为具有在poly(A)尾巴中掺入非A修饰功能的蛋白质、基因等。所述可在不含poly(A)尾巴的RNA末端连接含有非 A修饰的poly(A)尾巴的物质可为具有在RNA末端连接含有非A修饰的poly(A)尾巴功能的蛋白质、基因等，也可为具有在RNA末端连接含有非A修饰的poly(A)尾巴功能的蛋白质、基因等与含有非A修饰poly(A)尾巴的组合物。
上述应用中，所述转录模板可为PCR双链DNA产物、酶切双链DNA产物、质粒、黏粒、噬菌体或病毒载体。
上述应用中，所述poly(A)尾巴非A修饰可为poly(A)尾巴中G、C和/或U的插入。即，含有非A修饰的poly(A)尾巴为非纯A序列。
上述应用中，所述促进poly(A)尾巴非A修饰的物质可为蛋白质或生物材料，所述蛋白质为如下A1)-A5)中的任一种：
A1)非经典的PAP(ncPAP)；
A2)GLD-4、TENT4A或TENT4B蛋白质；
A3)氨基酸序列是SEQ ID No.2的GLD-4蛋白质；
A4)将序列表中SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/ 或缺失和/或添加且具有相同功能的蛋白质；
A5)在A1)或A2)或A3)或A4)的N端或/和C端连接标签得到的融合蛋白质；
所述生物材料为下述B1)至B5)中的任一种：
B1)编码所述蛋白质的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子的细胞系、或含有B2)所述表达盒的细胞系。
上述应用中，A2)所述蛋白质可为与GLD-4蛋白质具有75％或75％以上同一性且具有相同功能的蛋白质。所述具有75％或75％以上同一性为具有75％、具有80％、具有85％、具有90％、具有95％、具有96％、具有97％、具有98％或具有99％的同一性。
上述应用中，B1)所述核酸分子可为如下b11)或b12)或b13)或b14)：
b11)编码序列是序列表中SEQ ID No.1的cDNA分子或DNA分子；
b12)序列表中SEQ ID No.1所示的DNA分子；
b13)与b11)或b12)限定的核苷酸序列具有75％或75％以上同一性，且编码所述蛋白质的cDNA分子或DNA分子；
b14)在严格条件下与b11)或b12)或b13)限定的核苷酸序列杂交，且编码所述蛋白质的cDNA分子或DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码所述蛋白质的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明的所述蛋白质的核苷酸序列75％或者更高同一性的核苷酸，只要编码所述蛋白质且具有所述蛋白质功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码SEQ ID No.2所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述应用中，所述严格条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5MNaPO4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS 中漂洗；还可为：50℃，在7％SDS、0.5M NaPO4和1mM EDTA的混合溶液中杂交，在 50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO4和1mM EDTA 的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、 0.5M NaPO4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交,然后用2×SSC,0.1％SDS和1×SSC,0.1％ SDS各洗膜一次；也可为：2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次 5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；也可为：0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述应用中，B2)所述的含有编码所述蛋白质的核酸分子的表达盒(基因表达盒)，是指能够在宿主细胞中表达所述蛋白质的DNA，该DNA不但可包括启动所述蛋白质编码基因转录的启动子，还可包括终止所述蛋白质编码基因基因转录的终止子。进一步，所述表达盒还可包括增强子序列。
可用现有的表达载体构建含有所述蛋白质编码基因表达盒的重组载体。
上述应用中，所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述应用中，所述微生物可为酵母、细菌、藻或真菌。
上述应用中，所述细胞系可包括也可不包括繁殖材料。
上述应用中，所述mRNA翻译可为真核生物中的mRNA翻译。
上述应用中，所述mRNA翻译可为所述真核生物生殖细胞或体细胞中的mRNA翻译。
上述应用中，所述真核生物可为动物或植物或真核微生物。
所述动物可为哺乳动物(如人、小鼠、兔或猪)或非哺乳动物(如斑马鱼、果蝇或线虫)。
所述植物可为双子叶植物(如拟南芥)或单子叶植物(如水稻)。
所述真核微生物可为酵母。
本发明还提供了提高mRNA翻译效率的方法，所述方法包括：对目的mRNA的poly(A)尾巴进行非A修饰，实现目的mRNA翻译效率的提高。
上述方法中，对目的mRNA的poly(A)尾巴进行非A修饰可通过直接化学合成掺入非A 修饰完成，也可通过生物化学方法在mRNA末端生成含有非A修饰的poly(A)尾巴完成，也可通过在mRNA末端连接含有非A修饰的poly(A)尾巴完成，也可通过将包括含有非A修饰的poly(A)尾巴模板的mRNA转录模板进行转录完成。
上述方法中，所述非A修饰通过施加所述促进poly(A)尾巴非A修饰的物质实现。
上述方法中，所述mRNA翻译可为真核生物中的mRNA翻译。
上述方法中，所述mRNA翻译可为所述真核生物生殖细胞或体细胞中的mRNA翻译。
上述方法中，所述真核生物可为动物或植物或真核微生物。
所述动物可为哺乳动物(如人、小鼠、兔或猪)或非哺乳动物(如斑马鱼、果蝇或线虫)。
所述植物可为双子叶植物(如拟南芥)或单子叶植物(如水稻)。
所述真核微生物可为酵母。
本发明还提供了提高mRNA翻译效率的产品，所述产品为所述促进poly(A)尾巴非A修饰的物质。
上述产品中，所述mRNA翻译可为真核生物中的mRNA翻译。
上述产品中，所述mRNA翻译可为所述真核生物生殖细胞或体细胞中的mRNA翻译。
上述产品中，所述真核生物可为动物或植物或真核微生物。
所述动物可为哺乳动物(如人、小鼠、兔或猪)或非哺乳动物(如斑马鱼、果蝇或线虫)。
所述植物可为双子叶植物(如拟南芥)或单子叶植物(如水稻)。
所述真核微生物可为酵母。
本发明中，poly(A)尾巴的非A修饰是指poly(A)尾巴中含有G、C和/或U。poly(A)尾巴的非A修饰可通过将poly(A)尾巴中任意一个或多个A替换为G、C和/或U，或在poly(A) 尾巴中任意位置添加一个或多个G、C和/或U实现。“多个”可为两个或三个或四个或五个或六个或七个或八个或九个或十个等。
本发明中poly(A)尾巴中非A修饰的G、C和/或U的位置与个数没有要求，只要可以提高翻译效率，均在本发明的保护范围内。
在本发明的一个实施例中，含有非A修饰的poly(A)尾巴的序列为SEQ ID No.4、5、6。
本发明通过分析人、小鼠、兔、猪、斑马鱼、果蝇、线虫、拟南芥、水稻和酵母的poly(A) 尾巴，发现pATIM在真核生物中是RNA poly(A)尾巴的一种保守特性，其分布没有物种和组织特异性。通过分析mRNA的翻译，发现pATIM可以提高翻译效率。遗传学分析显示，线虫中的一种非典型poly(A)聚合酶GLD-4可通过建立pATIM来调控翻译。并通过在体外转录合成的mRNA的poly(A)尾巴中添加非A修饰，与相应的含有纯A的同样长度poly(A)尾巴的mRNA进行比较，进一步发现poly(A)尾巴中非A修饰可以有效地促进mRNA的翻译，poly(A)尾巴中的G、C、U的添加可以促进mRNA的翻译并提高相应蛋白质的含量。说明，poly(A)尾巴中非A修饰在真核生物中扮演的潜在普遍性翻译调控角色，更证实了poly(A)尾巴中非A修饰是提高mRNA效率的有效工具。
近些年来，医药工程领域基于mRNA的相关治疗方法蓬勃发展(Sahin et al.,2014)。由于mRNA更易设计和制备，与其他基于蛋白的疗法相比，mRNA疗法的成本优势十分突出。 mRNA疫苗的临床前开发可以十分迅速，对于应对突发性新发传染病往往具有巨大潜力 (Deweerdt,2019；Zhong et al.,2018)。第一种mRNA疫苗从开始研发到进入1/2期临床试验只花了1个月(Pardi et al.,2018；Thanh Le et al.,2020；Wang et al.,2020)。在mRNA疗法和疫苗研发过程中，mRNA翻译效率的提高是一大主要技术挑战(Sahin et al.,2014；Zhang et al.,2019)。poly(A)尾巴中非A修饰促进翻译效率新机制的发现，可以使得在mRNA的poly(A)尾巴中掺入非A修饰作为一种全新的技术进一步推动 mRNA疗法、疫苗等多种mRNA应用。
附图说明
图1为poly(A)尾巴序列分析流程图。
图2为poly(A)尾巴非A修饰与翻译效率正相关。
(A-D)不同样品中全基因组范围内poly(A)尾巴具有/不具有poly(A)尾巴非A修饰的基因翻译效率(mRNA-normalized TE)累积分布频率(CDF)。作图数据的来源样品分别为：(A)GV 期小鼠卵子(基因数：有G，n＝4104；无G，n＝1363；有C，n＝4109；无C，n＝1358；有U，n＝4116；无U，n＝1351)，(B)线虫生殖细胞(基因数：有G，n＝1390；无G，n＝1585；有C，n＝2233；无C，n＝742；有U，n＝2170；无U，n＝805)，(C)线虫体细胞(基因数：有G，n＝539；无G，n＝934；有C，n＝909；无C，n＝564；有U，n＝842；无U，n＝631)，(D)HeLa细胞(基因数：有G，n＝2075；无G，n＝810；有C，n＝2323；无C，n＝562；有U，n＝1884；无U，n＝1001)。 poly(A)尾巴非A修饰和TE之间的相关性用Kolmogorov-Smirnov(KS)检验计算p值。
(E-F)具有/不具有poly(A)尾巴非A修饰的基因质谱定量蛋白丰度累积分布频率图。数据分别来自于GV期卵子(E)(基因数：有G，n＝2307；无G，n＝768；有C，n＝2308；无C， n＝767；有U，n＝2306；无U，n＝769)，和HeLa细胞(F)(基因数：有G，n＝2398；无G，n＝1029；有C，n＝2694；无C，n＝733；有U，n＝2166；无U，n＝1261)。p值由KS检验求得。
有G：pATIM-G，即poly(A)尾巴中含有G；
无G：poly(A)尾巴中不含G；
有C：pATIM-C，即poly(A)尾巴中含有C；
无C：poly(A)尾巴中不含C；
有U：pATIM-U，即poly(A)尾巴中含有U；
无U：poly(A)尾巴中不含U。
TE：翻译效率(Translational efficiency)。
P值是统计两条累计分布曲线的KS统计检验p值。
图3为10个物种的GLD-4的同源基因所编码蛋白质的序列分析。下划线表示对于催化活性和NTP结合重要的氨基酸。
图4为GLD-4是线虫里poly(A)尾巴非A修饰的建立和翻译效率的调控所必需的。
(A)箱型图分别展示了WT和gld-4(ef15)突变体背景下，每个基因含有pATIM-G(左)， pATIM-C(中)和pATIM-U(右)修饰的转录本占比。依据转录本含有poly(A)尾巴非A修饰比例的低(在WT中一个基因包含的转录本含有G,C,U修饰的转录本数量占总的有poly(A)尾巴转录本数比例小于5％)或者高(在WT中一个基因包含的转录本含有G,C,U修饰的转录本数量占总的有poly(A)尾巴转录本数比例大于等于5％)，WT中的基因被分为2组(基因数：低G，n＝2758；高G，n＝169；低C，n＝2374；高C，n＝553；低U，n＝2392；高U，n＝535)。 p值由双尾t检验求得。
(B)gld-4 RNAi(gld-4突变体)和WT线虫中mRNA校正的核糖体结合(TE)变化累积频率分布图。数据分别展示了具有及不具有poly(A)尾巴非A修饰基因的数据(基因数：有G，n＝970；无G，n＝2869；有C，n＝1873；无C，n＝1966；有U，n＝1816；无U，n＝2023)。P值是统计两条累计分布曲线的KS统计检验p值。
图5为poly(A)尾巴非A修饰增强了翻译效率。
(A)尾巴长度一致但具有不同poly(A)尾巴非A修饰的报告mRNA示意图。
(B)基于不同poly(A)尾巴的转录本翻译效率报告系统示意图。
(C)HeLa细胞转染不同poly(A)尾巴报告mRNA18小时后的图像。标尺长度：100μm。DIC表示微分干涉对比明场成像。
(D)HeLa细胞转染报告mRNA42小时后表达的FACS定量结果。上图为荧光信号散点图，下图为荧光信号分布直方图。
(E)HeLa细胞转染报告mRNA42h后的qRT-PCR(左，mRNA水平)和免疫印迹(右，蛋白水平，使用抗Flag抗体检测所有报告基因融合表达的Flag标签)定量结果。
图6为不同时间点报告mRNA的表达定量分析。
(A)FACS(流式细胞荧光分选技术)检测到的转染后不同时间点HeLa细胞中报告EGFPmRNA表达水平定量分析。对照组为未转染mRNA的细胞。A98和A98pATIMs-2为分别转染了具有A98和A98pATIMs-2尾巴报告mRNA的细胞。
(B)FACS检测到的转染后不同时间点HeLa细胞中EGFP的相对荧光强度的定量分析。参与分析的细胞与上述(A)中的细胞一致。
P2表示较低严谨度的阈值门，包括荧光较弱的细胞；P3表示较高严谨度的阈值门，只包括荧光强的细胞。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量实验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应 DNA/RNA的5′末端核苷酸，末位均为相应DNA/RNA的3′末端核苷酸。
下述实施例中的gld-4(ef15)突变体(Mark Schmid,BeateKüchler,Christian R.Eckmann,Two conserved regulatory cytoplasmic poly(A)polymerases,GLD-4and GLD-2,regulate meiotic progression in C.elegans,Genes&Dev.2009.23:824-836)，公众可从申请人处获得该生物材料，该生物材料只为重复本发明的相关实验所用，不可作为其它用途使用。gld-4(ef15)突变体与背景个体的区别仅在于GLD-4基因发生了突变。 GLD-4基因的序列为序列表中SEQ ID No.1，编码SEQ ID No.2所示的GLD-4蛋白质。
实施例1、poly(A)尾巴非A修饰是真核生物RNA poly(A)尾巴的一个保守特征
本实施例通过分析人、小鼠、兔、猪、斑马鱼、果蝇、线虫、拟南芥、水稻和酵母的poly(A)尾巴，发现poly(A)尾巴非A修饰(简称为pATIM)在真核生物中是RNA poly(A) 尾巴的一种保守特性，其分布没有物种和组织特异性。
待测样本：人Hela细胞，小鼠肝脏，兔肝脏，猪肝脏，斑马鱼个体，果蝇个体，线虫个体，拟南芥，水稻叶片，酵母。
提取各样本的总RNA，而后制备PAIso-seq文库，然后进行第三代测序并对测序结果进行分析，将PAIso-seq测序序列比对到基因组，对于RNA末端非基因组模板转录的不能比对到基因组的序列即为poly(A)尾巴，详细分析方法见文献 (10.1038/s41467-019-13228-9)，步骤如图1所示，各物种的poly(A)尾巴见表1。
表1、10个物种的poly(A)尾巴非A修饰
实施例2、poly(A)尾巴非A修饰与翻译效率正相关
本实施例发明人利用已有的小鼠GV期卵子(GV卵)(GSE135525)、线虫生殖细胞(GSE62859)与体细胞(GSE58918)以及HeLa细胞(GSE79664)的核糖体结合数据来分析翻译效率与poly(A)尾巴非A修饰之间的关系，比较含有和不含有poly(A)尾巴非A修饰两组基因的翻译效率累计曲线。所分析基因为数据中至少检测到10条poly(A)尾巴的基因。在小鼠GV期卵子数据中一个基因“有G”定义为该基因的数据中大于等于25％的poly(A) 尾巴中有G，“无G”定义为该基因的数据中小于25％的poly(A)尾巴中无G，“有C”定义为该基因的数据中大于等于25％的poly(A)尾巴中有C，“无C”定义为该基因的数据中小于25％的poly(A)尾巴中无C，“有U”定义为该基因的数据中大于等于25％的poly(A)尾巴中有U，“无U”定义为该基因的数据中小于25％的poly(A)尾巴中无U。在线虫细胞和HeLa 细胞数据中，由于数据测序深度较低且非A碱基含量较GV卵中更低，一个基因“有G”定义为该基因的数据中至少有一条poly(A)尾巴中有G，“无G”定义为该基因的数据中所有的poly(A)尾巴中无G，“有C”定义为该基因的数据中至少有一条poly(A)尾巴中有C，“无C”定义为该基因的数据中所有的poly(A)尾巴中无C，“有U”定义为该基因的数据中至少有一条poly(A)尾巴中有U，“无U”定义为该基因的数据中所有的poly(A)尾巴中无U。
发现pATIM-G(即poly(A)尾巴中含有G)和pATIM-C(即poly(A)尾巴中含有C)修饰与翻译效率呈正相关。其中，翻译效率(Translational Efficiency,TE)是指样本中某个基因的总mRNA分子中与核糖体结合并进行翻译mRNA分子占总mRNA分子的比例，利用基因的Ribo-seq和RNA-seq的数据，就可以直接计算这个数值，具体计算公式为：TE ＝(FPKM inRibo-seq)/(FPKM in RNA-seq)。
结果发现，无论在生殖细胞还是体细胞中，pATIM-G和pATIM-C修饰都有显著更高的翻译效率(图2中A-D)。而pATIM-U(即poly(A)尾巴中含有U)修饰也可以提高翻译效率。
考虑到mRNA翻译的最终结果是相应蛋白的积累，发明人利用已发表的质谱蛋白组数据在GV卵(即小鼠GV期卵子)和HeLa细胞里分析了poly(A)尾巴非A修饰与蛋白丰度之间的关系，比较含有和不含有poly(A)尾巴非A修饰两组基因的翻译效率累计曲线。已发表的质谱蛋白组数据来自于：Bekker-Jensen et al.,2017；Wang et al.,2010 (10.1016/j.cels.2017.05.009；10.1073/pnas.1013185107)。
结果显示，无论是在GV卵里还是在HeLa细胞里，含有poly(A)尾巴非A修饰的基因均有着较高的表达丰度，这一结果也与核糖体结合数据相吻合(图2中E,F)。
上述系列结果显示，无论是在生殖细胞还是在体细胞中，poly(A)尾巴中的poly(A)尾巴非A修饰都可能对mRNA起到一种普遍的翻译调控作用，进一步表明poly(A)尾巴并非仅仅是mRNA的一个简单结构组成，更可以在生殖细胞和体细胞里作为mRNA功能的关键调控因子发挥作用。
实施例3、线虫中GLD-4通过建立poly(A)尾巴非A修饰增强翻译效率
Poly(A)聚合酶(PAP)催化产生了RNA poly(A)尾巴。经典PAP催化了转录偶联的mRNA 多聚腺苷酸化，而非经典的PAP(ncPAP)催化了转录后的poly(A)尾巴重新多聚腺苷酸化(Lim et al.,2014；Lim et al.,2018)。线虫里的两种ncPAP——全身性表达的GLD-2和主要表达于生殖细胞里的GLD-4对于生殖细胞发育十分重要(Schmid et al.,2009；Wanget al.,2002)。gld-2敲除可到导致poly(A)尾巴长度发生全局性降低；而gld-4敲除后，转录组水平的poly(A)尾巴长度仅有微弱下降(Nousch et al.,2014)。gld-4突变体中的总体翻译效率发生下降，而类似的下降并未在gld-2突变体中观察到(Millonigg et al.,2014；Nousch et al.,2014)，暗示GLD-2和GLD-4可能具有不同的功能机制。
实施例1中所提及的10个物种均含有1-2个GLD-4的同源基因，这些基因编码的蛋白都具有保守的核心核苷酸转移酶结构域(图3)。哺乳动物基因组中的GLD-4同源基因为TENT4A/B。纯化的TENT4A和TENT4B蛋白可以催化产生主要由A组成的、同时掺入G、U和C 的poly(A)尾巴，这一结果也与在HeLa细胞中所观察到的混合尾巴相一致(Limetal.,2018，10.1126/science.aam5794)。
本实施例发现，线虫中的一种非典型poly(A)聚合酶GLD-4可通过建立poly(A)尾巴非A 修饰来调控翻译，GLD-4/TENT4A/TENT4B催化产生了RNA poly(A)尾巴非A修饰。
发明人首先以gld-4(ef15)突变体的完整个体为待测样本，提取其总RNA，而后按照实施例1的方法制备PAIso-seq文库，然后进行第三代测序并对RNA poly(A)尾巴序列进行了分析，实验包含2个生物学重复，并以野生型线虫(WT)为对照。线虫中Gld-4的cDNA 序列为序列表中SEQ ID No.1，编码序列表中SEQ ID No.2所示的Gld-4蛋白质。
结果发现，野生型(WT)转录本中带有poly(A)尾巴非A修饰的基因，在gld-4突变体中有显著的pATIM-G、pATIM-C和pATIM-U修饰的丢失(图4中A)。这一结果显示，GLD-4可以催化产生带有poly(A)尾巴非A修饰的RNA尾巴。
考虑到线虫生殖细胞基因中具有poly(A)尾巴非A修饰的转录本比例与翻译效率呈正相关，发明人还比较了gld-4敲除前后，具有和不具有poly(A)尾巴非A修饰的基因，它们的翻译效率有没有发生变化。
符合预期的是，gld-4突变体中，相较于不具有poly(A)尾巴非A修饰的基因，具有poly(A) 尾巴非A修饰的基因有显著更高的翻译效率(图4中B)，这一结果再次证实GLD-4可通过催化poly(A)尾巴非A修饰来提高翻译效率。
综上，GLD-4/TENT4A/TENT4B这类ncPAP能够催化产生poly(A)尾巴中的这种进化上保守的、可促进mRNA翻译的poly(A)尾巴非A修饰，提高mRNA的翻译效率。
实施例4、人工合成的poly(A)尾巴非A修饰可促进翻译
由于poly(A)尾巴非A修饰与翻译效率正相关，而线虫中GLD-4的丢失伴随着poly(A) 尾巴非A修饰的减少与翻译效率的降低，因而发明人进一步利用体外合成的mRNA报告系统来研究poly(A)尾巴非A修饰在翻译效率方面的作用。发明人合成了poly(A)尾巴长度相同，但poly(A)尾巴非A修饰数量不同的荧光报告基因(EGFP与mCherry)mRNA(图5中A)，并将这些mRNA转染HeLa细胞，检测对应的报告mRNA及其编码蛋白的动态变化(图5中B)。步骤如下：
所合成mRNA为四种EGFP的mRNA与四种mCherry的mRNA，EGFP的四种mRNA分别是EGFP 的CDS序列后分别跟随A98、A98pATIMs-1、A98pATIMs-2或A98pATIMs-3序列，mCherry的四种mRNA分别是mCherry的CDS序列后分别跟随A98、A98pATIMs-1、A98pATIMs-2或A98pATIMs-3序列。A98、A98pATIMs-1、A98pATIMs-2、A98pATIMs-3与CDS序列间均含有编码FLAG标签的RNA序列(GAUUACAAGGACGACGAUGACAAG)。
A98、A98pATIMs-1、A98pATIMs-2与A98pATIMs-3分别为四种poly(A)尾巴，其序列分别为序列表中SEQ ID No.3-6(即图5A中TGA之后的序列)，A98不含有poly(A)尾巴非A 修饰。EGFP的CDS序列为序列表中SEQ ID No.7(该序列为图5A中自5’端至TGA的序列)，mCherry的CDS序列为序列表中SEQ ID No.8(该序列为图5A中自5’端至TGA的序列)。
用RNase-free水溶解各mRNA，使用前储存于-80℃。
HeLa细胞培养于37℃、5％CO2的潮湿培养箱中，培养基为含有10％FBS(Gibco)的DMEM 培养基(Invitrogen)。HeLa细胞的mRNA转染使用的是Lipofectamine MessengerMAX转染试剂(Invitrogen)。每种mRNA的转染量均为2500ng/(250,000–1,000,000细胞)。
转染之后的特定时间点，拍照、进行FACS分析或者收取细胞进行下一步实验。
在转染18小时，荧光显微镜检测转染不同EGFPmRNA的细胞的荧光信号，结果显示，与纯A尾巴(A98)对照相比，具有poly(A)尾巴非A修饰尾巴的mRNA有显著更高的EGFP荧光蛋白表达水平(图5中C)。
在转染42小时，用荧光激活细胞分选技术(Fluorescent-activated CellSorting，FACS) 来比较A98pATIMs-2修饰的poly(A)尾巴和纯A尾巴之间的翻译效率差异。相较之下， A98pATIMs-2报告基因的EGFP荧光有所增强；而在mCherry中也观察到了类似的荧光增强现象(图5中D)。这些结果显示poly(A)尾巴非A修饰对于翻译的增强效果并不局限于某种特定的mRNA。利用未转染mRNA的HeLa细胞作为对照组(Control)。
在转染42小时，分别通过免疫印迹和qPCR定量分析这些细胞中的蛋白和mRNA水平。利用未转染mRNA的HeLa细胞作为对照组(Control)。
qPCR定量分析所用引物如表2所示。
表2、引物序列
免疫印迹所用一抗：鼠抗FLAG(Sigma,F1804；1:1000稀释)、鼠抗Tubulin(Sigma,T9026；1:1000稀释)。二抗：荧光抗体(LI-COR,925-32210,1:10000稀释)。Tubulin 为内参。
与荧光显微镜镜检观察和FACS分析结果一致的是，转染了带有poly(A)尾巴非A修饰 mRNA的细胞，其Cherry和EGFP蛋白产物均有更高的积累(图5中E)。qPCR结果显示，相应的mRNA水平并无增加，显示蛋白水平的增加是由更高的翻译效率而非更强的mRNA稳定性所导致的(图5中E)。
细胞转染后分时间点收样检测荧光，结果显示，mRNA的这种翻译增强效果在转染6-120h 内是持续的(图6)。利用未转染mRNA的HeLa细胞作为对照组(Control)。
上述实验结果清楚地揭示，poly(A)尾巴非A修饰可以极大地促进mRNA翻译。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
<110>中国科学院遗传与发育生物学研究所
<120>poly(A)尾巴非A修饰在促进mRNA翻译中的应用
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Ala Pro Ser His Lys His Arg Asn Ala His Pro Gln Arg Gln Arg Pro
645 650 655
Ser Ile Arg Asn Leu Ser Gln Gly Ser Asp Gly Ser Asp Glu Tyr Asn
660 665 670
Val Glu Ser Trp Asn Asn Asn Ile Arg Gln Gly Arg Arg Ala Ser Ser
675 680 685
Asn Ser Pro Ser Pro Ser Arg Gln Gln Thr Asn Thr Arg Asn Cys Gly
690 695 700
Pro Thr Asn Asn Ile Pro Tyr Asp Ser Phe Arg Ser Gln Asn Lys Asn
705 710 715 720
Ser Thr Leu Asp Gly Ser Asn Asn Ser Ser Glu Glu Pro Ile Thr Met
725 730 735
Tyr Ala Asp Val Val Lys Lys Lys Ser Ser Ile Thr Thr Ser Thr Asn
740 745 750
Thr Ser Thr Ala Asp Val Asn Val Thr Asn Gly Asn Pro Ile Pro Ala
755 760 765
Asn Gly Ile Ile Pro Gln Ser Met Ala Val Val Asn Val Gly Arg Gly
770 775 780
Ser Tyr Arg Asn Ala Leu Thr Thr Ser Pro Met Thr Pro Pro Ser Ala
785 790 795 800
His Thr Ser Met Gln Lys Gln His His Leu Arg Lys Asp Asn Glu Cys
805 810 815
Gly Phe Asp Asn Asn Ser Ala Thr Ser Ser Thr Asp Leu Ser His His
820 825 830
Gln Pro Gln Leu Val Pro Pro Val Asn Arg Leu Gln Arg
835 840 845
<210> 3
<211> 98
<212> RNA
<213> 人工序列（Artificial sequence）
<400> 3
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 60
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 98
<210> 4
<211> 98
<212> RNA
<213> 人工序列（Artificial sequence）
<400> 4
aaaaaaaaaaaaaaaaaaaacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaauaaaaaaa 60
aaaaaaaacaaaaaaaaaaaaaaaaaaaaaaagaaaaa 98
<210> 5
<211> 98
<212> RNA
<213> 人工序列（Artificial sequence）
<400> 5
aaaaaaaaaaaacaaaaaaaaaaaaaaaaaaaagaaaaaaaaaaaaaaaaaaaaaaaaac 60
ccaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaaaaaa 98
<210> 6
<211> 98
<212> RNA
<213> 人工序列（Artificial sequence）
<400> 6
aaaaaaaaaaaaacaaaaaaaaaaaaaaaaaaaggaaaaaaaaaaaaaaaaaaaaaaaaa 60
aaaagaaaaaaaaaaaaaaaaaaaaaaaaaccaaaaaa 98
<210> 7
<211> 717
<212> RNA
<213> 人工序列（Artificial sequence）
<400> 7
auggugagcaagggcgaggagcuguucaccgggguggugcccauccuggucgagcuggac 60
ggcgacguaaacggccacaaguucagcguguccggcgagggcgagggcgaugccaccuac 120
ggcaagcugacccugaaguucaucugcaccaccggcaagcugcccgugcccuggcccacc 180
cucgugaccacccugaccuacggcgugcagugcuucagccgcuaccccgaccacaugaag 240
cagcacgacuucuucaaguccgccaugcccgaaggcuacguccaggagcgcaccaucuuc 300
uucaaggacgacggcaacuacaagacccgcgccgaggugaaguucgagggcgacacccug 360
gugaaccgcaucgagcugaagggcaucgacuucaaggaggacggcaacauccuggggcac 420
aagcuggaguacaacuacaacagccacaacgucuauaucauggccgacaagcagaagaac 480
ggcaucaaggugaacuucaagauccgccacaacaucgaggacggcagcgugcagcucgcc 540
gaccacuaccagcagaacacccccaucggcgacggccccgugcugcugcccgacaaccac 600
uaccugagcacccaguccgcccugagcaaagaccccaacgagaagcgcgaucacaugguc 660
cugcuggaguucgugaccgccgccgggaucacucucggcauggacgagcuguacaag 717
<210> 8
<211> 708
<212> RNA
<213> 人工序列（Artificial sequence）
<400> 8
auggugagcaagggcgaggaggauaacauggccaucaucaaggaguucaugcgcuucaag 60
gugcacauggagggcuccgugaacggccacgaguucgagaucgagggcgagggcgagggc 120
cgccccuacgagggcacccagaccgccaagcugaaggugaccaaggguggcccccugccc 180
uucgccugggacauccuguccccucaguucauguacggcuccaaggccuacgugaagcac 240
cccgccgacauccccgacuacuugaagcuguccuuccccgagggcuucaagugggagcgc 300
gugaugaacuucgaggacggcggcguggugaccgugacccaggacuccucccugcaggac 360
ggcgaguucaucuacaaggugaagcugcgcggcaccaacuuccccuccgacggccccgua 420
augcagaagaagaccaugggcugggaggccuccuccgagcggauguaccccgaggacggc 480
gcccugaagggcgagaucaagcagaggcugaagcugaaggacggcggccacuacgacgcu 540
gaggucaagaccaccuacaaggccaagaagcccgugcagcugcccggcgccuacaacguc 600
aacaucaaguuggacaucaccucccacaacgaggacuacaccaucguggaacaguacgaa 660
cgcgccgagggccgccacuccaccggcggcauggacgagcuguacaag 708
Claims (1)
1.通过在poly(A)尾巴中掺入四处非A修饰在提高哺乳动物中mRNA的翻译效率中的应用；所述非A修饰为poly(A)尾巴中含有G、C和/或U；
其中，所述非A修饰的poly(A)尾巴的序列选自SEQ ID NO: 4、5和6。
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Citations (4)
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|---|---|---|---|---|
| US6054296A (en) * | 1988-10-24 | 2000-04-25 | Symbicom Ab | 66 kDa antigen from Borrelia |
| AU2011253592A1 (en) * | 2005-09-28 | 2011-12-15 | Biontech Ag | Modification of RNA, producing an increased transcript stability and translation efficiency |
| CN108823225A (zh) * | 2018-05-31 | 2018-11-16 | 中国科学院理化技术研究所杭州研究院 | 蛋白质翻译过程中直接实现脂肪酸修饰的表达系统及应用 |
| CN110218753A (zh) * | 2019-05-27 | 2019-09-10 | 湖北爱济莱斯生物科技有限公司 | 一种提高体外合成mRNA稳定性的方法 |
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| DE102005046490A1 (de) * | 2005-09-28 | 2007-03-29 | Johannes-Gutenberg-Universität Mainz | Modifikationen von RNA, die zu einer erhöhten Transkriptstabilität und Translationseffizienz führen |
| US11926819B2 (en) * | 2019-05-28 | 2024-03-12 | The Regents Of The University Of California | Methods of adding polymers to ribonucleic acids |
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| US6054296A (en) * | 1988-10-24 | 2000-04-25 | Symbicom Ab | 66 kDa antigen from Borrelia |
| AU2011253592A1 (en) * | 2005-09-28 | 2011-12-15 | Biontech Ag | Modification of RNA, producing an increased transcript stability and translation efficiency |
| CN108823225A (zh) * | 2018-05-31 | 2018-11-16 | 中国科学院理化技术研究所杭州研究院 | 蛋白质翻译过程中直接实现脂肪酸修饰的表达系统及应用 |
| CN110218753A (zh) * | 2019-05-27 | 2019-09-10 | 湖北爱济莱斯生物科技有限公司 | 一种提高体外合成mRNA稳定性的方法 |
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| WO2023029600A1 (zh) | 2023-03-09 |
| CN115216483A (zh) | 2022-10-21 |
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