Figure 3

Functional affects associated with RBM10 variant expression. (A) In GLC20 cells, comparative expression levels of RBM10v1 and RBM10v2 transcripts encoding the valine-retaining and valine-lacking isoforms, as determined by RNA-seq. *p < 0.05. (B) NUMB alternative splicing in A549, HeLa, BEAS-2B and GLC20 cells. (i) and (ii) are 2% agarose gels with SYBR®safe, showing representative amplicon expression levels following end-point PCR with (i) 28 cycles, or (ii) 40 cycles, using NUMB exon 11-spanning primers and (i) 19 cycles or (ii) 25 cycles for GAPDH. (iii) Following densitometry of amplicons from six end-point PCR reactions (three at 28 cycles and three at 40 cycles) of the two different cDNA preparations from one RNA extraction for each cell line, the percentage of the NUMB exon 11 exclusion variant was calculated and plotted. Error bars represent the standard error of the mean. Significances were calculated using an unpaired Student’s t-test, with *p < 0.05, **p < 0.01 and ****p < 0.0001. (C) Model depicting how the +/− valine isoforms of RBM10v1 might influence NUMB exon 11 alternative splicing. The plus and minus valine isoforms are present, at low levels, in normal cells, and contribute to the production of both the exon 11 inclusion and exclusion variants of NUMB. Both RBM10 isoforms are able to bind NUMB pre-mRNA, but the minus valine isoform of RBM10v1 does so with higher affinity, having the classical α-helix structure. The plus valine isoform does not bind as efficiently, thereby interfering with recognition of the intron 10 3′splice site, resulting in NUMB exon 11 exclusion.