Identification of transcripts harboring premature termination codons by NMD inhibition in a panel of CCRCC cell lines

Identification of transcripts harbouring premature termination codons by NMD inhibition (GINI method) in a panel of clear cell renal cell carcinoma cell lines. Sporadic clear cell renal cell carcinoma (cRCC) is genetically characterized by the recurrent loss of chromosome 3p, with a hotspot for copy number loss in the 3p21 region. In this study, we applied a method called Gene Identification by Nonsense Mediated mRNA decay Inhibition (GINI) on a panel of 10 cRCC cell lines with 3p21 copy number loss to identify biallelic inactivated genes located at 3p21. This analysis revealed inactivation of the histone methyltransferase gene SETD2, located on 3p21.31, as a common event in cRCC cells. SETD2 is nonredundantly responsible for trimethylation of the histone mark H3K36. Consistent with this function, we observed loss or decrease of H3K36me3 in 7 out of 10 cRCC cell lines. Identification of missense mutations in 2 of 10 primary cRCC tumor samples further supported the involvement of loss of SETD2 function in the development of cRCC tumors. GINI (Gene identification by Nonsense mediated mRNA decay Inhibition) was applied to a panel of 10 clear cell renal cell carcinoma cell lines. Cells were treated with emetine, caffeine or were not treated. Experiments were performed in duplo (biological replicates). Gene expression was measured in two proximal tubular epithelial cell lines (in duplo, biological replicates) to assess expression in the normal situation. Two different matrixes are shown: one that contains the data from the GINI experiment, the other contains the transcript levels in untreated cRCC and PTEC cells.