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Supplementary Methods, Figures 1-10, Tables 1-19 from Global Changes in Processing of mRNA 3′ Untranslated Regions Characterize Clinically Distinct Cancer Subtypes

Molecular cancer diagnostics are an important clinical advance in cancer management, but new methods are still needed. In this context, gene expression signatures obtained by microarray represent a useful molecular diagnostic. Here, we describe novel probe-level microarray analyses that reveal connections between mRNA processing and neoplasia in multiple tumor types, with diagnostic potential. We now show that characteristic differences in mRNA processing, primarily in the 3′-untranslated region, define molecular signatures that can distinguish similar tumor subtypes with different survival characteristics, with at least 74% accuracy. Using a mouse model of B-cell leukemia/lymphoma, we find that differences in transcript isoform abundance are likely due to both alternative polyadenylation (APA) and differential degradation. While truncation of the 3′-UTR is the most common observed pattern, genes with elongated transcripts were also observed, and distinct groups of affected genes are found in related but distinct tumor types. Genes with elongated transcripts are overrepresented in ontology categories related to cell-cell adhesion and morphology. Analysis of microarray data from human primary tumor samples revealed similar phenomena. Western blot analysis of selected proteins confirms that changes in the 3′-UTR can correlate with changes in protein expression. Our work suggests that alternative mRNA processing, particularly APA, can be a powerful molecular biomarker with prognostic potential. Finally, these findings provide insights into the molecular mechanisms of gene deregulation in tumorigenesis. [Cancer Res 2009;69(24):9422–30]

Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and immunoglobulin class switching in activated B cells. Because AID has no known target-site specificity, there have been efforts to identify non-immunoglobulin AID targets. We show here that AID acts promiscuously, generating widespread DNA double-strand breaks (DSBs), genomic instability and cytotoxicity in B cells with less homologous recombination ability. We demonstrate that the homologous-recombination factor XRCC2 suppressed AID-induced off-target DSBs, promoting B cell survival. Finally, we suggest that aberrations that affect human chromosome 7q36, including XRCC2, correlate with genomic instability in B cell cancers. Our findings demonstrate that AID has promiscuous genomic DSB-inducing activity, identify homologous recombination as a safeguard against off-target AID action, and have implications for genomic instability in B cell cancers.

Previous quantitative trait locus (QTL) analysis of an intercross involving the inbred mouse strains NZB/BlNJ and SM/J revealed QTL for a variety of complex traits. Many QTL have large intervals containing hundreds of genes, and methods are needed to rapidly sort through these genes for probable candidates. We chose nine QTL: the three most significant for high-density lipoprotein (HDL) cholesterol, gallstone formation, and obesity. We searched for candidate genes using three different approaches: mRNA microarray gene expression technology to assess >45,000 transcripts, publicly available SNPs to locate genes that are not identical by descent and that contain nonsynonymous coding differences, and a mass-spectrometry-based proteomics technology to interrogate nearly 1000 proteins for differential expression in the liver of the two parental inbred strains. This systematic approach reduced the number of candidate genes within each QTL from hundreds to a manageable list. Each of the three approaches selected candidates that the other two approaches missed. For example, candidate genes such as Apoa2 and Acads had differential protein levels although the mRNA levels were similar. We conclude that all three approaches are important and that focusing on a single approach such as mRNA expression may fail to identify a QTL gene.