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

Abstract

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.

RNA-Sequencing Analysis of 5' Capped RNAs Identifies Many New Differentially Expressed Genes in Acute Hepatitis C Virus Infection

“…It will be of interest to determine the similarities and differences in gene expression of acutely HCV infected proliferative and growth arrested differentiated human hepatoma-derived cells [36]. In comparison with our reported ENCODE tilling array (interrogates 1% of the human genome) analysis of HCV infected cirrhotic liver that identified 95 differentially expressed genes as compared to controls, our current analysis of acutely infected hepatoma cells showed that 20% of these genes were also differentially expressed [14]. This is a relatively large percentage of overlap since liver represents a much more complex transcriptome due to the presences of a number of other cell types besides hepatocytes and the fact that the ENCODE analysis was of chronically infected cirrhotic liver.…”

“…We have reported previously that the analysis of 5' capped RNA, rather than poly(A) selected RNA, provides a more sensitive means of detecting differentially expressed genes in HCV infected biospecimens [14]. Our approach purifies Pol II transcripts by binding their 5' caps with a high-affinity variant of the RNA cap binding protein (eIF4E K119A ), enabling us to isolate and study transcripts with short or missing 3' poly(A) ends [12,13,15,16,17].…”

Gene Signature in Sessile Serrated Polyps Identifies Colon Cancer Subtype

“…Total RNA was isolated using TRIzol (Invitrogen) and quality of RNA assessed by an Agilent 2000 bioanalyzer as described previously (21,23,24). RNA sequencing was performed on 86 individual colon samples: 21 SSA/Ps (12 syndromic and 9 sporadic), 10 hyperplastic polyps, 10 adenomatous polyps, 21 uninvolved colon, 20 control colon, and 4 colon cancer samples.…”

TRIM22: A Diverse and Dynamic Antiviral Protein

“…Moreover, TRIM22 expression is significantly upregulated during clearance of hepatitis C virus (HCV) in chimpanzees [52]. These findings are paralleled in human infections, as TRIM22 is significantly upregulated in cirrhotic liver from HCV patients and patients with mild chronic HCV infection and no fibrosis [53]. Further research is needed to assess the role of TRIM22 in inhibiting HBV and HCV in vivo .…”