Ring1B has a remarkable genomic structure.

<p>The genomic structure of Ring1B is depicted in the upper pane, the approximate sizes of exons in the middle pane and introns in the lower pane, as well as the translational start and stop codons. Exon 7 greatly varies in length, sometimes measuring up to 3,5 kb (A). Apart from a 12 nucleotide stretch in the middle, which is only seen in human and pig Ring1B, IRESes of different mammals are virtually identical. The exact mouse sequence depicted, comprising the entire IRES until the translational start, is used in all our assays (B). A 5′ RLM-RACE was performed to determine the main transcriptional start (C). In lane 5 a 100 bp marker was loaded, with the most prominent band being 600 bp. In ES cells (lane 1), primary MEFs (lane 2) and 293 cells (lane 3) the most abundant fragment is approximately 380 bp long. This suggests a transcriptional start at approximately 154 bp 5′ of the 3′ splice site of the first exon, which was verified after sequencing the fragments. Lane 4 shows an internal control PCR, using the same 3′ Ring1B inner primer, but with a Ring1B 5′ primer instead of an adapter primer. The Ring1B 5′ primer is located downstream of the translational start, so this PCR serves as an internal control for RNA quality and primer specificity.