ENCODE Tiling Array Analysis Identifies Differentially Expressed Annotated and Novel 5′ Capped RNAs in Hepatitis C Infected Liver

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.

慢性丙型肝炎病毒（HCV）感染的基因芯片（microarray）研究，已为宿主对病毒感染的应答提供了极具价值的信息。然而，近期针对人类转录组（transcriptome）的研究显示，基因组中此前未被注释的区域存在广泛转录现象，且众多RNA转录本的3′端聚腺苷酸（poly(A)）尾较短或缺失该结构。我们提出假设：结合使用ENCODE平铺阵列芯片（ENCODE tiling arrays，覆盖1%基因组）与通过RNA聚合酶II（Pol II）独特的5′端m7GpppN帽结构亲和纯化其转录本的方法，可鉴定出丙型肝炎病毒（HCV）感染过程中肝脏内差异表达的、此前未被报道的注释基因与未注释基因。研究人员采用ENCODE平铺阵列芯片，对5′端带帽（5′-capped）与聚腺苷酸化阳性（poly(A)+）两类RNA群体进行了分析。与对照肝脏相比，HCV相关肝硬化肝脏中共有64个注释基因的表达量显著上调；其中27个基因（占比42%）仅通过分析5′端带帽RNA得以鉴定。另有31个注释基因的表达量显著下调，其中16个基因（占比50%）仅通过分析5′端带帽RNA得以鉴定。生物信息学分析结果显示，带帽RNA的分析结果一致性更高，可提供内含子区域更全面的表达谱，并能在HCV相关肝硬化肝脏的基因组未注释区域中鉴定出上调的RNA聚合酶II转录活性区域。其中两个区域已通过聚合酶链式反应（PCR）与快速扩增cDNA末端（RACE）分析得到验证。对肝脏活检标本的实时定量PCR（qPCR）分析显示，在伴有轻度炎症且无纤维化的丙型肝炎患者样本中，这些未注释转录本以及IRF1、TRIM22与MET基因的表达同样出现上调。结合ENCODE平铺阵列芯片与5′端带帽RNA的分析策略，可获取更多基因表达相关信息，并能鉴定出此前在HCV感染肝脏中未被报道的新型上调RNA聚合酶II转录本。该策略，尤其是与新型RNA测序技术结合使用时，还可用于进一步明确特定疾病状态下差异调控的RNA聚合酶II转录本，以及研究由前体mRNA剪接或3′端聚腺苷酸化状态改变所调控的RNA分子。