Figure 4

AS-NMD within the TH1L gene. A Histogram representing QPCR validation results for TH1L. Bars represent mean fold change in mRNA levels in response to either UPF1 knockdown (left panel, N = 8) or cycloheximide treatment (right panel, N = 8) ± SEM. p-value summary (Student's t test, one tail): * p < 0.05, ** p < 0.01, *** p < 0.001. QPCR primers were located upstream of the schematic shown in B, in a region expected to be unaffected by alternative splicing. B. Upper panel: schematic of the AS-NMD event within 3' UTR of TH1L produced using the UCSC genome browser [114], running 5' left to 3' right. Boxes represent exons while chevroned lines represent introns. The upper blue cartoon indicates the Refseq annotated 3' UTR structure, the thinning of the box indicating the end of the protein coding sequence. The underlying cartoons are Genbank mRNAs illustrating the AS isoforms predicted by our analysis, the red mRNA represents the NMD sensitive isoform - a retained intron is spliced to make the normal stop codon appear premature. The lower blue histogram represents conservation across 17 vertebrate species as calculated by [115]. Lower panel: RT-PCR of the AS-NMD event, illustrating the effect of UPF1 knockdown. M: 1 kb plus marker (GE healthcare). RT-: addition of RT performed without reverse transcriptase. PCR-: PCR performed without template. Underlying numbers indicate the mean (± SEM, N = 3) percentage of the total signal from both bands represented by the NMD-sensitive for each siRNA treatment. C. UCSC genome browser schematic and RT-PCR of a second predicted event within TH1L. Use of an alternative 5' splice site results in a frameshift creating a downstream PTC. The arrangement of elements is the same as for section B.