Chimeric RNA

Abstract: RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Abstract: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare liver tumor affecting adolescents and young adults with no history of primary liver disease or cirrhosis. We identified a chimeric transcript that is expressed in FL-HCC but not in adjacent normal liver and that arises as the result of a ~400-kilobase deletion on chromosome 19. The chimeric RNA is predicted to code for a protein containing the amino-terminal domain of DNAJB1, a homolog of the molecular chaperone DNAJ, fused in frame with PRKACA, the catalytic domain of protein kinase A. Immunoprecipitation and Western blot analyses confirmed that the chimeric protein is expressed in tumor tissue, and a cell culture assay indicated that it retains kinase activity. Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100% of the FL-HCCs examined (15/15) suggests that this genetic alteration contributes to tumor pathogenesis.

Abstract: A recombinant plasmid has been constructed for the expression of inserted DNA sequences coding for polypeptide chains using the simian virus 40 early promoter and splicing and polyadenylylation signals from the rabbit beta-globin gene. The coding regions for two prokaryotic methotrexate-resistant dihydrofolate reductases were introduced into the expression vector. When mouse fibroblasts were exposed to these recombinant plasmids, it was possible to select methotrexate-resistant clones that had integrated the plasmids and produced a chimeric RNA coding for the prokaryotic enzyme.

Abstract: Chromosomal rearrangements that create gene fusions are common features of human tumors. The prevailing view is that the resultant chimeric transcripts and proteins are abnormal, tumor-specific products that provide tumor cells with a growth and/or survival advantage. We show that normal endometrial stromal cells contain a specific chimeric RNA joining 5' exons of the JAZF1 gene on chromosome 7p15 to 3' exons of the Polycomb group gene JJAZ1/SUZ12 on chromosome 17q11 and that this RNA is translated into JAZF1-JJAZ1, a protein with anti-apoptotic activity. The JAZF1-JJAZ1 RNA appears to arise from physiologically regulated trans-splicing between precursor messenger RNAs for JAZF1 and JJAZ1. The chimeric RNA and protein are identical to those produced from a gene fusion found in human endometrial stromal tumors. These observations suggest that certain gene fusions may be pro-neoplastic owing to constitutive expression of chimeric gene products normally generated by trans-splicing of RNAs in developing tissues.