Figure 3

Novel transcripts that encode abnormal hRasGRP4 isoforms in the PBMCs. The top panel shows the exon structure and corresponding functional motifs of hRasGRP4, which include the Ras exchange motif (REM), CDC25-like GEF domain (CDC25 box), calcium-binding EF hands, and diacylglycerol -binding C1 domain. Splice variants 5 to 14 in the lower panel correspond to the 10 new hRasGRP4 transcripts identified in our RA patients. If translated, the number of amino acids (AA) in each expressed isoform is indicated. Eighty hRasGRP4-specific cDNAs were isolated and sequenced from 16 healthy individuals. Two, 13, 1, 1 and 1 of these cDNAs corresponded to splice variants 1, 5, 6, 7, and 8 respectively. The remaining 62 sequenced cDNAs in this control group corresponded to the normal, full-length hRasGRP4 isoform. Ninety and 25 hRasGRP4-specific cDNAs were isolated and sequenced from 18 RA patients undergoing treatment and from five untreated RA patients, respectively. The type and number of the identified defective transcripts in these patients are shown. One hundred hRasGRP4 cDNAs from 20 patients with other autoimmune diseases were sequenced, resulting in 36 defective and 64 normal hRasGRP4 transcripts as shown. Variants 1, 9, 12, and 14 possess a premature translation-termination codon (*). Intron 1 is 3.9 kb and many of the other introns in this approximately 17.2 kb human gene are > 1 kb. Because the PCR approach used to identify and quantitate the new isoforms would miss an isoform that contains intron 1, it is likely that additional splice variants exist in PBMCs that contain one or more large introns.