ENCODE tiling array analysis identifies differentially expressed annotated and novel 5' capped RNAs in hepatitis C infected liver.
ENCODE tiling array analysis identifies differentially expressed annotated and novel 5' capped RNAs in hepatitis C infected liver.
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DOI：
10.1371/journal.pone.0014697
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发表时间：
2011-02-16
期刊：
影响因子：
3.7
通讯作者：
Hagedorn CH
中科院分区：
文献类型：
--
作者：
Folkers ME;Delker DA;Maxwell CI;Nelson CA;Schwartz JJ;Nix DA;Hagedorn CH
慢性丙型肝炎感染的微阵列研究提供了关于宿主对病毒感染的反应的有价值的信息。然而，最近对人类转录组的研究表明，在以前未注释的基因组区域中普遍存在转录，并且许多RNA转录物具有短的或缺乏3′ poly（A）末端。我们假设，使用ENCODE平铺阵列（基因组的1%）结合亲和纯化Pol II RNA（通过其独特的5′ m7 GpppN帽），将识别出丙型肝炎病毒（HCV）感染期间肝脏中差异表达的先前未描述的注释和未注释基因。使用ENCODE平铺阵列分析5′-加帽和poly（A）+RNA群体。与对照组相比，HCV感染组中有64个注释基因显著增加;其中27个（42%）基因仅通过分析5′加帽RNA鉴定。31个注释基因显著减少;其中16个（50%）仅通过分析5′加帽RNA鉴定。生物信息学分析表明，加帽的RNA产生更一致的结果，提供了一个更广泛的内含子区域的表达谱，并确定上调Pol II转录活性区在HCV肝硬化基因组的未注释区域。其中两个区域通过PCR和RACE分析得到验证。肝活检标本的qPCR分析表明，这些未注释的转录本以及IRF 1、TRIM 22和MET在轻度炎症和无纤维化的丙型肝炎中也上调。5′加帽RNA结合ENCODE平铺阵列的分析提供了额外的基因表达信息，并鉴定了先前在HCV感染的肝脏中未描述的新型上调的Pol II转录物。这种方法，特别是当与新的RNA测序技术相结合时，也应该有助于进一步确定在特定疾病状态下差异调节的Pol II转录物，以及研究由前mRNA剪接或3′聚腺苷酸化状态变化调节的RNA。
Microarray studies of chronic hepatitis C infection have provided valuable information regarding the host response to viral infection. However, recent studies of the human transcriptome indicate pervasive transcription in previously unannotated regions of the genome and that many RNA transcripts have short or lack 3′ poly(A) ends. We hypothesized that using ENCODE tiling arrays (1% of the genome) in combination with affinity purifying Pol II RNAs by their unique 5′ m7GpppN cap would identify previously undescribed annotated and unannotated genes that are differentially expressed in liver during hepatitis C virus (HCV) infection. Both 5′-capped and poly(A)+ populations of RNA were analyzed using ENCODE tiling arrays. Sixty-four annotated genes were significantly increased in HCV cirrhotic as compared to control liver; twenty-seven (42%) of these genes were identified only by analyzing 5′ capped RNA. Thirty-one annotated genes were significantly decreased; sixteen (50%) of these were identified only by analyzing 5′ capped RNA. Bioinformatic analysis showed that capped RNA produced more consistent results, provided a more extensive expression profile of intronic regions and identified upregulated Pol II transcriptionally active regions in unannotated areas of the genome in HCV cirrhotic liver. Two of these regions were verified by PCR and RACE analysis. qPCR analysis of liver biopsy specimens demonstrated that these unannotated transcripts, as well as IRF1, TRIM22 and MET, were also upregulated in hepatitis C with mild inflammation and no fibrosis. The analysis of 5′ capped RNA in combination with ENCODE tiling arrays provides additional gene expression information and identifies novel upregulated Pol II transcripts not previously described in HCV infected liver. This approach, particularly when combined with new RNA sequencing technologies, should also be useful in further defining Pol II transcripts differentially regulated in specific disease states and in studying RNAs regulated by changes in pre-mRNA splicing or 3′ polyadenylation status.