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Quantification of Spatiotemporal Patterns of Ras Isoform Expression During Development

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Quantification of Spatiotemporal Patterns of Ras Isoform Expression During Development

Anna U Newlaczyl et al. Sci Rep.

Abstract

Ras proteins are important signalling hubs frequently dysregulated in cancer and in a group of developmental disorders called Rasopathies. Three Ras genes encode four proteins that differentially contribute to these phenotypes. Using quantitative real-time PCR (qRT-PCR) we have measured the gene expression profiles of each of the Ras isoforms in a panel of mouse tissues derived from a full developmental time course spanning embryogenesis through to adulthood. In most tissues and developmental stages we observe a relative contribution of KRas4B > > NRas ≥ KRas4A > HRas to total Ras expression with KRas4B typically representing 60-99% of all Ras transcripts. KRas4A is the most dynamically regulated Ras isoform with significant up-regulation of expression observed pre-term in stomach, intestine, kidney and heart. The expression patterns assist interpretation of the essential role of KRas in development and the preponderance of KRas mutations in cancer.

Figures

Figure 1
Figure 1. Specific PCR amplification of Ras isoforms.
(A) Schematic of exon organization of Ras isoforms with coding areas in white and non-coding in black. Arrows indicate locations of isoform-specific Ras primer pairs. For KRas4B, the reverse primer was designed to span the exon3/4B splice boundary. (B) Ras isoform-specific primers were used in end-point PCR reactions with the indicated linearised templates of each Ras isoform plasmid. Products of the correct size were observed and there was no evidence of cross-amplification.
Figure 2
Figure 2. Quantitation of Ras isoform expression in adult tissues.
(A) Total Ras expression summed from qRT-PCR quantitation of HRas, KRas4A, KRas4B, NRas and ERas. (B) Analysis of the relative expression of Ras isoforms indicates that KRas4B represents 70–99% of total tissue Ras mRNA transcripts. Histogram employs log scale to show contribution of minor isoforms. Pie charts show the relative expression of all Ras isoforms except KRas4B. (C) Contribution of KRas4A and KRas4B to total KRas expression. n = 3 adult mice per tissue, n = 3 R1 mESC cell samples.
Figure 3
Figure 3. Ras isoform expression during development.
(A) Schematic of tissue collection schedule during developmental stages. (B) qRT-PCR quantitation of Ras isoform expression in mouse tissues. (C) Comparison of expression at P30 with E11.5 reveals dynamic changes amongst all Ras isoforms across tissues with most showing a decrease in Ras expression during maturation. Tukey plots depict data minima and maxima, the range between the first and third quartiles and the line indicates the median.

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