Alternative polyadenylation events in epithelial cells sense endometritis progression in dairy cows

Endometritis(inflammation of the endometrial lining) is one of the most devastating reproductive diseases in dairy cattle, resulting in substantial production loss and causing more than $650 million in lost revenue annually in the USA.We hypothesize that alternative polyadenylation(APA) sites serve as decisive sensors for endometrium health and disease in dairy cows. Endometrial cells collected from 18 cows with purulent vaginal discharge scored 0 to 2 were used for APA profiling with our whole transcriptome termini site sequencing(WTTS-seq) method. Overall, pathogens trigger hosts to use more differentially expressed APA(DE-APA), more intronic DE-APA, more DE-APA sites per gene and more DE-genes associated with inflammation. Host CD59 molecule(CD59), Fc fragment of IgG receptor IIa(FCGR2A), lymphocyte antigen 75(LY75) and plasminogen(PLG) may serve as initial contacts or combats with pathogens on cell surface, followed by activation of nuclear receptor subfamily 1 group H member 4(NR1H4) to regulate AXL receptor tyrosine kinase(AXL), FGR proto-oncogene, Src family tyrosine kinase(FGR), HCK protooncogene, Src family tyrosine kinase(HCK) and integrin subunit beta 2(ITGB2) for anti-inflammation. This study is the first to show significance of cilium pathways in endometrium health and animal reproduction. MIR21 and MIR30A would be perfect antagonistic biomarkers for diagnosis of either inflammation or anti-inflammation. These novel findings will set precedent for future genomic studies to aid the dairy industry develop new strategies to reduce endometritis incidence and improve fertility.

Alternative polyadenylation-related genetic variants contribute to bladder cancer risk

Aberrant alternative polyadenylation(APA)events play an important role in cancers,but little is known about whether APA-related genetic variants contribute to the susceptibility to bladder cancer.Previous genome-wide association study performed APA quantitative trait loci(apaQTL)analyses in bladder cancer,and identified 17955 single nucleotide polymorphisms(SNPs).We found that gene symbols of APA affected by apaQTL-associated SNPs were closely correlated with cancer signaling pathways,high mutational burden,and immune infiltration.Association analysis showed that apaQTL-associated SNPs rs34402449 C>A,rs2683524 C>T,and rs11540872 C>G were significantly associated with susceptibility to bladder cancer(rs34402449:OR=1.355,95%confidence interval[CI]:1.159-1.583,P=1.33×10^(−4);rs2683524:OR=1.378,95%CI:1.164-1.632,P=2.03×10^(−4);rs11540872:OR=1.472,95%CI:1.193-1.815,P=3.06×10^(−4)).Cumulative effect analysis showed that the number of risk genotypes and smoking status were significantly associated with an increased risk of bladder cancer(P_(trend)=2.87×10^(−12)).We found that PRR13,being demonstrated the most significant effect on cell proliferation in bladder cancer cell lines,was more highly expressed in bladder cancer tissues than in adjacent normal tissues.Moreover,the rs2683524 T allele was correlated with shorter 3′untranslated regions of PRR13 and increased PRR13 expression levels.Collectively,our findings have provided informative apaQTL resources and insights into the regulatory mechanisms linking apaQTL-associated variants to bladder cancer risk.

Alternative polyadenylation:An untapped source for prostate cancer biomarkers and therapeutic targets?

Objective:To review alternative polyadenylation(APA)as a mechanism of gene regulation and consider potential roles for APA in prostate cancer(PCa)biology and treatment.Methods:An extensive review of mRNA polyadenylation,APA,and PCa literature was performed.This review article introduces APA and its association with human disease,outlines the mechanisms and components of APA,reviews APA in cancer biology,and considers whether APA may contribute to PCa progression and/or produce novel biomarkers and therapeutic targets for PCa.Results:Eukaryotic mRNA 30-end cleavage and polyadenylation play a critical role in gene expression.Most human genes encode more than one polyadenylation signal,and produce more than one transcript isoform,through APA.Polyadenylation can occur throughout the gene body to generate transcripts with differing 30-termini and coding sequence.Differences in 30-untranslated regions length can modify post-transcriptional gene regulation by microRNAs and RNA binding proteins,and alter mRNA stability,translation efficiency,and subcellular localization.Distinctive APA patterns are associated with human diseases,tissue origins,and changes in cellular proliferation rate and differentiation state.APA events may therefore generate unique mRNA biomarkers or therapeutic targets in certain cancer types or phenotypic states.Conclusions:The full extent of cancer-associated and tissue-specific APA events have yet to be defined,and the mechanisms and functional consequences of APA in cancer remain incompletely understood.There is evidence that APA is active in PCa,and that it may be an untapped resource for PCa biomarkers or therapeutic targets.

中华蜜蜂泛素基因及其全长转录本的发掘及分析

旨在利用三代Nanopore测序技术发掘中华蜜蜂(Apis cerana cerana)泛素(ubiquitin)基因及其全长转录本。基于前期获得的中华蜜蜂工蜂4~6日龄幼虫肠道的全长转录组数据,使用BLAST工具将全长转录本的序列比对到KEGG和Nr数据库以鉴定泛素基因及其全长转录本。采用Astalavista软件分析泛素基因的可变剪接(alternative splicing,AS)事件,并通过RT-PCR加以验证。通过TAPIS pipeline软件分析泛素基因的可变多聚腺苷酸化(alternativepolyadenylation,APA)位点。共鉴定到48个中华蜜蜂泛素基因和435条泛素基因相关全长转录本。共发掘到48个泛素基因的74次AS事件,包括31次内含子保留事件,19次可变3′端剪接事件,16次可变5′端剪接事件及8次外显子互斥事件。RT-PCR结果证实了3种AS事件类型的真实性。共预测到38个泛素基因含有1个及以上的APA位点,并且在APA位点的上游鉴定到多个motif,一致性序列为:KCWYTDYTMWSYG MWSCARAWCCAG AATGAYCCWYWGGHWVMWGWDRTRGC。研究结果丰富了中华蜜蜂泛素基因及其全长转录本信息,为持续深入开展相关功能研究提供参考和依据。

中华蜜蜂酚氧化酶和丝氨酸蛋白酶相关基因及其全长转录本发掘与分析

为深入开展中华蜜蜂相关研究提供参考信息和基础,利用已获得的纳米孔(Nanopore)测序数据对中华蜜蜂Apis cerana cerana的酚氧化酶(phenoloxidase,PO)和丝氨酸蛋白酶(serine protease,SP)相关基因和全长转录本进行发掘和分析。通过Blast工具将中华蜜蜂所有全长转录本比对KEGG和Nr数据库以筛选出PO和SP相关基因和全长转录本。采用gffcompare软件将PO和SP相关全长转录本与东方蜜蜂参考基因组(ACSNU-2.0)已注释基因进行比较以优化结构。使用Astalavista软件鉴定PO和SP相关基因的可变剪接(alternative splicing,AS)事件,再利用IGV浏览器进行结构可视化。通过PCR验证AS事件的真实性。利用TAPIS pipeline预测和分析可变多聚腺苷酸化(alternative polyadenylation,APA)位点,并通过TBtool软件鉴定APA位点上游的基序(motif)。鉴定到中华蜜蜂PO和SP相关的42个基因与146条转录本。优化了16个参考基因组已注释PO和SP相关基因的结构,其中5′端延长和3′端延长的基因均有7个,5′端和3′端同时延长的基因有2个。鉴定到PO和SP相关的10个基因的389次AS事件,其中最丰富的AS类型是5′端可变剪接。PCR结果证实了2次AS事件的真实性。共鉴定到34个PO和SP相关基因含有1个及以上的APA位点,其中含有3个APA位点的基因数量最多;在APA位点上游鉴定到多个motif,一致性序列为:GGHKSYWSHHTRATWTCNBHDMRRYWYRTNYTVACNGCKGCDCAYTGYR。鉴定到中华蜜蜂PO和SP相关的42个基因和146条全长转录本,优化了东方蜜蜂参考基因组已注释的PO和SP相关基因结构,并发掘出PO和SP相关基因的389次AS事件和237个APA位点。

中华蜜蜂细胞吞噬与包囊作用相关基因全长转录本鉴定及分析

【目的】系统鉴定和分析中华蜜蜂(Apis cerana cerana)吞噬与包囊作用相关基因和全长转录本,为深入开展相关基因和剪接体的功能研究奠定基础。【方法】基于前期已获得的高质量中华蜜蜂纳米孔长读段测序数据,通过Blast工具将全长转录本比对Nr数据库筛选出吞噬与包囊作用相关基因和全长转录本。利用gffcompare软件将全长转录本与东方蜜蜂(Apis cerana)参考基因组上注释的转录本进行比较,鉴定未注释的新基因和新转录本。利用TAPIS pipeline预测和分析吞噬与包囊作用相关基因的可变多聚腺苷酸化(alternative polyadenylation, APA)位点,并通过TBtools软件鉴定APA位点上游的基序(motif)。使用Astalavista软件鉴定可变剪接(alternative splicing, AS)事件,并通过IGV浏览器进行结构可视化。通过RT-PCR验证AS事件的真实性。【结果】共鉴定到中华蜜蜂吞噬与包囊作用相关的基因66个和全长转录本395条,发掘出东方蜜蜂参考基因组未注释的2个新基因和303条新转录本。对参考基因组已注释的34个基因进行了结构优化,分别延伸了18个基因的5′端和12个基因的3′端,同时延长了4个基因的5′端和3′端。共鉴定到含有1个及以上APA位点的吞噬与包囊作用相关基因47个,其中多于5个APA位点的基因最多,为32个。在APA位点上游鉴定到多个基序,一致性序列为:GRBGCNKSDAACAAYTRBGCBMRNGGBYAYTAYWCNVWNGG。共鉴定到吞噬与包囊作用相关基因的AS事件296次,其中包括131次可变3′端剪接(alternative 3′splice site, A3SS)、85次内含子保留(intron retention, IR)、70次可变5′端剪接(alternative 5′splice site, A5SS)和10次外显子跳跃(exon skipping, ES)。RT-PCR结果显示,扩增的目的片段大小符合预期,证实了随机选择的2次AS事件的真实性。【结论】系统鉴定了中华蜜蜂吞噬与包囊作用相关基因和全长转录本以及AS事件和APA位点,优化了东方蜜蜂参考基因组注释的吞噬与包囊作用相关基因的结构。

mRNA alternative polyadenylation (APA) in regulation of gene expression and diseases

The mRNA polyadenylation plays essential function in regulation of mRNA metabolism.Mis-regulations of mRNA polyadenylation are frequently linked with aberrant gene expression and disease progression.Under the action of polyadenylate polymerase,poly(A)tail is synthesized after the polyadenylation signal(PAS)sites on the mRNAs.Alternative polyadenylation(APA)often occurs in mRNAs with multiple poly(A)sites,producing different 3'ends for transcript variants,and therefore plays important functions in gene expression regulation.In this review,we first summarize the classical process of mRNA 3'-terminal formation and discuss the length control mechanisms of poly(A)innucleus and cytoplasm.Thenwe review the research progress on alternative polyadenylation regulation and the APA site selection mechanism.Finally,we summarize the functional roles of APA in the regulation of gene expression and diseases including cancers.

SIZ1-mediated SUMOylation of CPSF100 promotes plant thermomorphogenesis by controlling alternative polyadenylation

Under warm temperatures,plants adjust their morphologies for environmental adaption via precise gene expression regulation.However,the function and regulation of alternative polyadenylation(APA),an important fine-tuning of gene expression,remains unknown in plant thermomorphogenesis.In this study,we found that SUMOylation,a critical post-translational modification,is induced by a long-term treat-ment at warm temperatures via a SUMO ligase SIZ1 in Arabidopsis.Disruption of SIZ1 altered the global usage of polyadenylation signals and affected the APA dynamic of thermomorphogenesis-related genes.CPSF100,a key subunit of the CPSF complex for polyadenylation regulation,is SUMOylated by SIZ1.Importantly,we demonstrated that SUMOylation is essential for the function of CPSF1oo in genome-wide polyadenylation site choice during thermomorphogenesis.Further analyses revealed that the SUMO conjugation on CPSF100 attenuates its interaction with two isoforms of its partner CPSF30,increasing the nuclear accumulation of CPsF1oo for polyadenylation regulation.In summary,our study uncovers a regulatory mechanism of APA via SiZ1-mediated SUMOylation in plant thermomorpho-genesis.

Disruption of PABPN1 phase separation by SNRPD2 drives colorectal cancer cell proliferation and migration through promoting alternative polyadenylation of CTNNBIP1

Generally shortened 3′UTR due to alternative polyadenylation(APA)is widely observed in cancer,but its regulation mechanisms for cancer are not well characterized.Here,with profiling of APA in colorectal cancer tissues and poly(A)signal editing,we firstly identified that the shortened 3′UTR of CTNNIBP1 in colorectal cancer promotes cell proliferation and migration.We found that liquid-liquid phase separation(LLPS)of PABPN1 is reduced albeit with higher expression in cancer,and the reduction of LLPS leads to the shortened 3′UTR of CTNNBIP1and promotes cell proliferation and migration.Notably,the splicing factor SNRPD2 upregulated in colorectal cancer,can interact with glutamic-proline(EP)domain of PABPN1,and then disrupt LLPS of PABPN1,which attenuates the repression effect of PABPN1 on the proximal poly(A)sites.Our results firstly reveal a new regulation mechanism of APA by disruption of LLPS of PABPN1,suggesting that regulation of APA by interfering LLPS of 3′end processing factor may have the potential as a new way for the treatment of cancer.

基于3'RACE的胃癌细胞肿瘤相关基因的APA位点分析

目的分析胃癌细胞中肿瘤相关基因的APA(alternative polyadenylation)位点变化。方法我们选取肿瘤相关基因HSP90α和SEC11A,利用3'RACE方法在胃癌细胞系MKN45、MKN28和AGS中扩增其mRNA的3'端序列,经过测序,与已知数据库UCSC比对分析其APA位点变化。结果与正常数据库相比,胃癌细胞中HSP90α和SEC11A两个基因均出现新的APA位点,产生含有不同长度的3'UTR(untranslated region)的新mRNA异构体。结论胃癌细胞中HSP90α和SEC11A的APA位点发生变化,产生新的mRNA异构体,为研究肿瘤发生发展提供了新思路。

stAPAminer:Mining Spatial Patterns of Alternative Polyadenylation for Spatially Resolved Transcriptomic Studies

Alternative polyadenylation(APA)contributes to transcriptome complexity and gene expression regulation and has been implicated in various cellular processes and diseases.Singlecell RNA sequencing(scRNA-seq)has enabled the profiling of APA at the single-cell level;however,the spatial information of cells is not preserved in scRNA-seq.Alternatively,spatial transcriptomics(ST)technologies provide opportunities to decipher the spatial context of the transcriptomic landscape.Pioneering studies have revealed potential spatially variable genes and/or splice isoforms;however,the pattern of APA usage in spatial contexts remains unappreciated.In this study,we developed a toolkit called stAPAminer for mining spatial patterns of APA from spatially barcoded ST data.APA sites were identified and quantified from the ST data.In particular,an imputation model based on the k-nearest neighbors algorithm was designed to recover APA signals,and then APA genes with spatial patterns of APA usage variation were identified.By analyzing wellestablished ST data of the mouse olfactory bulb(MOB),we presented a detailed view of spatial APA usage across morphological layers of the MOB.We compiled a comprehensive list of genes with spatial APA dynamics and obtained several major spatial expression patterns that represent spatial APA dynamics in different morphological layers.By extending this analysis to two additional replicates of the MOB ST data,we observed that the spatial APA patterns of several genes were reproducible among replicates.stAPAminer employs the power of ST to explore the transcriptional atlas of spatial APA patterns with spatial resolution.This toolkit is available at https://github.com/BMILAB/stAPAminer and https://ngdc.cncb.ac.cn/biocode/tools/BT007320.

Stress responses of plants through transcriptome plasticity by mRNA alternative polyadenylation

The sessile nature of plants confines their responsiveness to changing environmental conditions.Gene expression regulation becomes a paramount mechanism for plants to adjust their physiological and morphological behaviors.Alternative polyadenylation(APA)is known for its capacity to augment transcriptome diversity and plasticity,thereby furnishing an additional set of tools for modulating gene expression.APA has also been demonstrated to exhibit intimate associations with plant stress responses.In this study,we review APA dynamic features and consequences in plants subjected to both biotic and abiotic stresses.These stresses include adverse environmental stresses,and pathogenic attacks,such as cadmium toxicity,high salt,hypoxia,oxidative stress,cold,heat shock,along with bacterial,fungal,and viral infections.We analyzed the overarching research framework employed to elucidate plant APA response and the alignment of polyadenylation site transitions with the modulation of gene expression levels within the ambit of each stress condition.We also proposed a general APA model where transacting factors,including poly(A)factors,epigenetic regulators,RNA m6A modification factors,and phase separation proteins,assume pivotal roles in APA related transcriptome plasticity during stress response in plants.

转录后选择性多聚腺苷酸化调控与糖尿病肾病

选择性多聚腺苷酸化(alternative polyadenylation,APA)作为重要的转录后调控机制,是真核细胞mRNA成熟过程中,由于不同多聚腺苷酸化信号位点的选择导致一个基因产生多个3’UTR序列长度不同的转录异构体,其中pre-mRNA序列中的顺式调控元件、对应的反式作用因子,例如细胞核中多种酶和蛋白因子等共同参与APA调控。研究发现,APA机制参与多种生理、病理进程,主要通过改变3’UTR序列长度影响对应反式作用因子的调控作用,进而调节mRNA定位、稳定性、翻译效率及蛋白的定位等。本文将系统阐述转录后APA的作用机制和研究现状,并结合新近研究进展,探讨APA在糖尿病肾病研究中的前景。

选择性多聚腺苷酸化在生物学过程中功能的研究进展

选择性多聚腺苷酸化(alternative polyadenylation,APA)在基因表达调控、发育、分化、转化和增殖过程中具有重要作用。文章综述了APA在细胞激活与增殖、发育和分化代谢调控中的功能,同时也综述了microRNAs和APA相互作用的调控网络关系,对了解APA对真核生物的生命活动具有重要的生物学意义。

mRNA成熟调控因子NUDT21对K562细胞转录本选择性剪接的影响

目的:研究mRNA前体3′端剪接调控因子NUDT21对白血病K562细胞转录本选择性剪接的影响。方法:以白血病K562细胞为研究对象,通过慢病毒敲减Nudix水解酶21(Nudix hydrolase 21,NUDT21)后,应用转录组测序RNA-Seq方法明确干扰前、后转录本的表达水平。生物信息学方法分析差异表达基因及可变剪接的变化,并用qPCR验证。结果:NUDT21敲减后K562细胞基因差异表达表现:上调5196条,下调3917条;转录本差异表达表现:上调22200条,下调19728条。通过GO和KEGG分析,NUDT21敲减后表达显著差异的转录本主要与细胞粘附分化、造血细胞系及自身免疫等过程相关。差异显著的选择性剪接共有436个,主要涉及细胞增殖、代谢等多个生物学过程的调控。ERBB2、MAPK激酶MKNK2、G蛋白偶联受体GRK6、真核细胞翻译延伸因子EEF1B2、细胞周期蛋白CCNL2、有丝分裂检查点蛋白BUB3等出现m RNA前体3′端的剪接改变。其中ERBB2mRNA变体1的表达减少,变体4的表达增多。结论:NUDT21通过包括mRNA前体3′端可变剪接在内的多种调控方式从较高层面影响细胞的生物学功能。

缢蛏多巴胺受体基因及其在损伤修复中的作用

多巴胺受体(dopamine receptor)作为生物体中重要的神经递质受体,在生物体的生长、发育、代谢等多个生理过程中都发挥着重要的功能。本研究从缢蛏Sinonovacula constricta转录组文库中筛选获得多巴胺受体基因的部分片段,结合RACE(rapid-amplification of cDNA ends)技术和降落PCR(polymerase chain reaction)技术,克隆得到缢蛏两个多巴胺受体的选择性多聚腺苷酸化(alternative polyadenylation,APA)的变异体,命名为ScDopR2a和ScDopR2b,长度分别为1824bp和2758bp。两个变异体均包含相同的5′非翻译区(untranslated regions,UTR)(24bp)和开放阅读框(open reading frame,ORF)(1440bp),共编码479个氨基酸残基。但是两个变异体的3′UTR长度不同,分别为360bp和1294bp,其中ScDopR2b在polyA尾前插入936bp。在同源的ORF区设计引物,采用实时定量PCR分析多巴胺受体基因在不同组织中的表达特征,结果表明多巴胺受体基因在水管、鳃、斧足的表达量显著高于其他组织。在该基因序列3′UTR插入片段区域设计特异引物检测ScDopR2b的组织表达情况,结果表明在水管、鳃、斧足的表达量仍高于其他组织,表达趋势与ORF区大致相同。进一步设计缢蛏组织损伤实验,对进水管前端进行损伤处理,在处理后的第4h、8h、12h、24h、48h、60h和72h取样,荧光定量检测结果显示,同源区表达量在12h和48h呈上调,在8h、24h和72h下调,且ScDopR2b表达趋势与同源区表达模式大致相同。研究结果表明,缢蛏多巴胺受体参与了损伤修复过程,其表达特征可能与多巴胺作为补偿性神经递质的作用有关。

选择性多聚腺苷酸化的生物学效应及其调控机制研究进展

真核生物基因的前体mRNA(pre-mRNA)及一些lncRNA在成熟过程中其3'端会发生剪切和多聚腺苷酸化反应(cleavage and polyadenylation, C/P),C/P的发生需要多聚腺苷酸化信号(polyadenylation signal, PAS)的存在。选择性多聚腺苷酸化(alternative cleavage and polyadenylation, APA)是指具有多个PAS的基因,在其mRNA3'端成熟过程中,由于选择不同的PAS,导致产生出多个3'UTR长度和序列组成不同的转录异构体。3'UTR长度和序列的不同会影响mRNA的稳定性、翻译效率、运输和细胞定位等,因此APA是真核生物的一个重要转录后调控方式。近年来,对大量动物、植物及酵母的基因组测序分析发现,APA在真核生物广泛存在,针对APA的生物学效应和调控机制开展了一系列研究。目前已鉴定出许多APA调控的顺式调控元件和反式作用因子。本文重点介绍了APA生物学效应和调控机制的最新研究进展,并探讨了未来APA调控的研究方向。

优雅蝈螽水溶性和类膜结合型海藻糖酶编码cDNA的鉴定及其在体内的表达（英文）

海藻糖酶是一种二糖水解酶,催化海藻糖转换为葡萄糖,为昆虫包括发育、壳多糖合成及飞翔代谢在内的多种生理过程所必需。尽管某些昆虫的海藻糖酶基因已被鉴定,但优雅蝈螽的海藻糖酶编码序列尚未见报告。本研究采用RACE结合多重PCR技术,分离鉴定优雅蝈螽的水溶性海藻糖酶(Gg Tre1)和类膜结合型海藻糖酶(Gg Tre2-like)的全长编码序列(cDNA),包括携带不同长度3'-非翻译区(3'-UTR)的3个Gg Tre1 cDNA亚型(Gen Bank:No.KY400001-KY400003)和3个Gg Tre2-like cDNA亚型(Gen Bank:No.KY400004-KY400006)。3个Gg Tre1 cDNA序列分别为2 107,2 021和1 914bp,具有相同长度的5'-UTR(33 bp),但3'-UTR长度不同,分别为322,248和129 bp。Gg Tre1-2 cDNA含1 740 bp,编码579个氨基酸残基组成的多肽链,分子量为67.29 k D;与之不同,根据cDNA演绎的Gg Tre1-1和Gg Tre1-3序列较Gg Tre1-2多4个氨基酸残基,多肽链的分子量为67.88 k D。3个Gg Tre2-like cDNA(Gg Tre2-like-1,-2和-3)序列全长分别为2 491,2 460和2 381 bp。5'-UTR均为284 bp,3'-UTR分别为398,367和285 bp。Gg Tre2-like cDNA开阅读框为1 809 bp,编码602氨基酸残基组成的多肽链,分子量为67.88 k D。实时定量PCR,分析Gg Tre1和Gg Tre2-like基因在雌、雄个体(各20个)的组织特异性表达。结果显示,Gg Tre1在卵巢和附腺表达量最高;Gg Tre2-like主要在卵巢表达,在雄性肌肉和马氏管的表达量高于其他组织。上述结果表明,本研究从优雅蝈螽分离到3'-UTR长度不同的3个水溶性和3个类膜结合型海藻糖酶cDNA序列。结果还提示,Gg Tre1在各组织的表达差异较大,而Gg Tre2-like在各组织的表达相对稳定。不同长度3'-UTR的Gg Tre1和Gg Tre2-like亚型的存在,以及不同长度的3'-UTR在翻译过程中的特殊作用,尚待今后研究证实。

Aberrant Alternative Polyadenylation is Responsible for Survivin Up-regulation in Ovarian Cancer

Background： Survivin is an oncoprotein silenced in normal mature tissues but reactivated in serous ovarian cancer （SOC）. Although transcriptional activation is assumed for its overexpression, the long 3＇-untranslated region （3＇-UTR） in survivin gene, which contains many alternate polyadenylation （APA） sites, implies a propensity for posttranscriptional control and therefore was the aim of our study. Methods： The abundance of the coding region, the proximal and the distal region of survivin mRNA 3＇-UTR, was evaluated by real-time polymerase chain reaction （PCR） in SOC samples, cell lines, and normal fallopian tube （NFT） tissues. The APA sites were confirmed by rapid amplification ofcDNA 3＇ ends and DNA sequencing. Real-time PCR were used to screen survivin-targeting microRNAs （miRNAs） that were inversely correlated with survivin. The expression of an inversely correlated miRNA was restored by pre-miRNA transfection or induction with a genotoxic agent to test its inhibitory effect on survivin overexpression. Results： Varying degrees of APA were observed in SOC by comparing the abundance of the proximal and the distal region of survivin 3＇-UTR, and changes of 3＇-UTR correlated significantly with survivin expression （r = 0.708, P 〈 0.01）. The main APA sites are proved at 1197 and 1673 of survivin 3＇-UTR by DNA sequencing. Higher level of 3＇-UTR proximal region than coding region was observed in NFT, as well as in SOC and cell lines. Among the survivin-targeting miRNAs, only a few highly expressed miRNAs were inversely correlated with survivin levels, and they mainly targeted the distal part of the 3＇-UTR. However, in ovarian cancer cells, restoration of an inversely correlated miRNA （miR-34c） showed little effect on survivin expression. Conclusions： In NFT tissues, survivin is not transcriptionally silenced but regulate posttranscriptionally. In SOC, aberrant APA leads to the shortening of survivin 3＇-UTR which enables it to escape the negative regulation of miRNAs and is responsible for survivin up-regulation.

CPSF30-L-mediated recognition of mRNA m^(6)A modification controls alternative polyadenylation of nitrate signaling-related gene transcripts in Arabidopsis

N6-methyladenosine(m^(6)A),a ubiquitous internal modification of eukaryotic mRNAs,plays a vital role in almost every aspect of mRNA metabolism.However,there is little evidence documenting the role of m^(6)A in regulating alternative polyadenylation(APA)in plants.APA is controlled by a large protein-RNA complex with many components,including CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30(CPSF30).In Arabidopsis,CPSF30 has two isoforms and the longer isoform(CPSF30-L)contains a YT512-B Homology(YTH)domain,which is unique to plants.In this study,we showed that CPSF30-L YTH domain binds to m^(6)A in v itro.In the cpsf30-2 mutant,the transcripts of many genes including several important nitrate signaling-related genes had shifts in polyadenylation sites that were correlated with m^(6)A peaks,indicating that these gene transcripts carrying m^(6)A tend to be regulated by APA.Wild-type CPSF30-L could rescue the defects in APA and nitrate metabolism in cpsf30-2,but m^(6)A-binding-defective mutants of CPSF30-L could not.Taken together,our results demonstrated that m^(6)A modification regulates APA in Arabidops is and revealed that the m^(6)A reader CPSF30-L affects nitrate signaling by controlling APA,shedding new light on the roles of the m^(6)A modification during RNA 3-end processing in nitrate metabolism.