Skip to main content
Figure 6 | BMC Genomics

Figure 6

From: Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay

Figure 6

AS-NMD within the HNRNPA2B1 gene. A. Histogram representing QPCR validation results for HNRNPA2B1. Bars represent mean fold change in mRNA levels in response to either UPF1 knockdown (left panel, N = 3 in this instance) 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. Schematic of the 3' UTR of HNRNPA2B1 produced using the UCSC genome browser [114], running 5' right to 3' left as indicated by the upper arrow. The upper black cartoon represents the prediction of the exonic structure (boxes) predicted by our bioinformatic analysis. The underlying red cartoons represent the highest scoring BLAT alignments [56] of several of the RACE sequences obtained in our analysis. Underlying these are cartoons representing the UCSC prediction of 3' UTR and the Refseq annotated UTR. The lower blue histogram represents conservation across 17 vertebrate species as calculated by [115]. The small UTR intron in the Refseq mRNA is too close to the stop codon for its splicing to make the stop codon appear premature (junction a, denoted by the green line). Splicing of the final intron in the UTR (junction b, denoted by the green line) would be expected to render all incumbent isoforms NMD sensitve. C. Histogram representing QPCR results for exon junctions a and b. Bars represent mean fold change (N = 3, ± SEM) in mRNA levels in response to UPF1 knockdown. D. Western blot of GFP positive HeLa cells sorted by flow cytometry. HeLa cells were co-transfected with a GFP expressing plasmid and either FLAG-tagged hnRNP A2 or an empty vector control. ERK1 was used as a loading control. E. Histogram representing QPCR results from parallel RNA samples to D. Bars represent mean fold change (N = 3, SEM) of HNRNPA2 and B1 in response to hnRNPA2 overexpression. p-value summary (Student's t test, two tails): * p < 0.05, ** p < 0.01, *** p < 0.001. F. Histogram representing QPCR results from parallel RNA samples to D. Bars represent mean fold change (N = 3, SEM) of UTR junctions a and b in response to hnRNPA2 overexpression. p-value summary (Student's t test, two tails): * p < 0.05, ** p < 0.01, *** p < 0.001.

Back to article page