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. 2005;12(2):69-81.
doi: 10.3727/000000005783992124.

Quantification of G protein Gaalphas subunit splice variants in different human tissues and cells using pyrosequencing

Ulrich H Frey  1 , Holger Nückel, Dobromir Dobrev, Iris Manthey, I E Sandalcioglu, Andreas Eisenhardt, Karl Worm, Hans Hauner, Winfried Siffert
Affiliations

Quantification of G protein Gaalphas subunit splice variants in different human tissues and cells using pyrosequencing

Ulrich H Frey et al. Gene Expr. 2005.

Abstract

The G protein Galphas is derived from four alternatively spliced transcripts, two long variants (Galphas(L)+CAG and Galphas(L)-CAG), which include an extra 45-bp segment, and two short variants (Galphas(S)+CAG and Galphas(S)-CAG). The long and short forms differ in each case by splicing in or out of a serine residue encoded at the 3' end of the variable exon 3. The relative expression of all four variants in human tissues is poorly investigated due to experimental limitations. We therefore established a method for reliable relative mRNA quantification of these splice variants based on the Pyrosequencing technology, and determined Galphas transcript ratios in various human tissues and cells. Galphas(S)/Galphas ratio was highest in blood mononuclear cells (0.84 +/- 0.02, n = 16) and lowest in the brain (0.51 +/- 0.14, n = 3). The different ranges resulted from differences in Galphas(S)+CAG ratios, which ranged from a total Galphas ratio of 0.32 +/- 0.07 (n = 12) in heart tissue to 0.57 +/- 0.03 (n = 16) in blood mononuclear cells (p < 0.0001), whereas the Galphas(S)-CAG ratio was rather constant and ranged from 0.22 +/- 0.04 (n = 7) in retinoblastoma cells to 0.27 +/- 0.04 in lymphocytes (p = 0.19). The Galphas(L)+CAG ratio ranged from 0.02 +/- 0.02 in heart tissue to 0.05 +/- 0.01 in retinoblastoma cells, with a varying proportion of Galphas(L)-CAG, which ranged from 0.14 +/- 0.02 in blood mononuclear cells to 0.41 +/- 0.08 in heart tissue. Stimulation of immortalized B lymphoblasts with isoproterenol resulted in significant changes of splice variant ratios. Our data indicate that changes of long and short ratios of Galphas in different tissues affected Galphas(L)-CAG and Gas(S)+CAG rather than Galphas(L)+CAG and Galphas(S-)CAG. Furthermore, stimulation of cells seemed to affect splice variant ratios. These results are, therefore, suggestive of different biological functions of these variants.

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Figures

Figure 1
Figure 1
Exon/intron organization map of exons 2–4 of human GNAS. Constitutively spliced exons (2 and 4) are represented by gray boxes and the alternatively spliced exon 3 by a black box. In addition to the consensus 3′ splice motif “AG” preceding exon 4, GNAS also has a noncanonical 3′ splice site (TG). Use of the TG 3′ splice site incorporates an additional CAG triplet into the spliced mRNA, resulting in an extra serine residue (white box). The four different mRNA species generated from alternative splicing are shown.
Figure 2
Figure 2
Discrimination of GαsL, GαsS+CAG, and GαsS−CAG variants. PCR primers were designed to span the alternatively spliced exon 3. The sequencing primer binds directly 5′ of exon 3 and detects exon 3 for GαsL as well as the additional CAG for GαsS+CAG and GαsS−CAG (upper left). The order in which each nucleotide was added into the Pyrosequencing reaction was chosen to yield single peaks in the pyrogram for certain splice variants. The numbers represent the quantity of nucleotides given by the sequence for the different variants (lower left). Plasmids pGEM-GαsL−CAG, pGEM-GαsS+CAG, and pGEM-GαsS−CAG were used to test if peak heights were comparable. The right side shows typical pyrograms resulting after sequencing of PCR products from plasmids. (A) pGEM-GαsL−CAG. (B) pGEM-GαsS+CAG. (C) pGEM-GαsS−CAG. GαsL−CAG can clearly be distinguished from the GαsS forms by producing a cytosine peak at position 5 while lacking a thymidine peak at position 6 (A). GαsS+CAG ratios can be calculated by subtracting cytosine at position 5 from cytosine at position 2 and comparing to thymidine at position 6, while GαsS−CAG ratios are calculated by subtracting the cytosine peak at position 2 from the guanosine peak at position 7 and comparing to thymidine at position 6 (B + C). All PCR reactions were carried out under the same conditions. GNAS_RT_Se2 and GNAS_RT_AS2_BT: PCR primers; GNAS_Pyr_Seq_spl: sequencing primer.
Figure 3
Figure 3
Discrimination of GαsL+CAG and GαsL−CAG. PCR sense primer was designed to align in exon 3, resulting in amplification of GαsL only (upper left). The order in which each nucleotide was added into the Pyrosequencing reaction was chosen to achieve unique peaks in the pyrogram for the different splice forms. The numbers represent the quantity of nucleotides given by the sequence for the different variants (lower left). Plasmids pGEM-GαsL+CAG and pGEM-GαsL−CAG were used to test if peak heights were comparable. The right side shows typical pyrograms resulting after sequencing of PCR products from plasmids. (A) pGEM-GαsL+CAG. (B) pGEM-GαsL−CAG. GαsL+CAG can clearly be distinguished from GαsL−CAG by producing a unique peak at cytosine 12 (A) while lacking a peak at guanine 13 (B). All PCR reactions were carried out under the same conditions. GNAS_RT_Se3 and GNAS_RT_AS2_BT: PCR primers; GNAS_Spl2_seq: sequencing primer.
Figure 4
Figure 4
Calibration plot for determination of ratios of Gαs using the Pyrosequencing method. (A) Plasmids containing full-length cDNA of GαsL−CAG and GαsS+CAG were mixed at different ratios in which C5 represents the long form and T6 the short form (Fig. 2). Plasmids were mixed as indicated and PCR was carried out with primers GNAS_RT_Se2 and GNAS_RT_AS2_BT. Products were visualized on a 2.5% agarose gel. Staining of the agarose gel with ethidium bromide resulted in brighter upper bands due to enhanced ethidium bromide intercalation into longer PCR products. (B) Sequencing was carried out using sequencing primer GNAS_Pyr_Seq_spl and peak heights were determined using Pyrosequencing software. Ratios were measured as (C5/G7) + (1 − T6/G7)]/2, with G7 representing peak height for both forms and plotted versus the expected ratios. (C) Plasmids containing full-length cDNA of GαsL−CAG and GαsL+CAG were mixed at different ratios in which C12 represents the +CAG form and G13 the −CAG form (Fig. 3). PCR was carried out with primers GNAS_RT_Se3 and GNAS_RT_AS2_BT. Ratios were measured as [(C12/G15) + (1 − G13/G15)]/2 with G15 representing peak height for both forms and plotted versus the expected ratios. For each data point two independent determinations were performed. A linear relationship between measured and calculated ratios over the whole range of tested ratios could be confirmed. Data are means ± SEM. r 2 = goodness of fit (values can range from 0.0 to 1.0; prediction of x values from y values is possible with a value close to 1.0).
Figure 5
Figure 5
Determination of GαsS/GαsL ratios from blood mononuclear cells and retinoblastoma cells. (A) Representative pyrogram of a CLL sample. PCR from cDNA from blood mononuclear cells was performed using primers GNAS_RT_SE2 and GNAS_RT_AS2_BT. Peak heights were determined using Pyrosequencing software. (B) Peak heights and calculations derived from CLL samples. Accuracy of the method was confirmed by comparing G7 (mean peak height 13.14 ± 0.90), which represents both isoforms and C5 + T6 (13.34 ± 0.85; p = 0.53 t-test), which represent the long and the short variants. (C) Agarose gel from three CLL and three retinoblastoma samples displaying different splicing patterns. Data are means ± SEM.
Figure 6
Figure 6
Absolute ratios of Gαs variants in different human tissues and cell lines. Ratios were calculated as follows with respect to Figures 2 and 3: GαsS+CAG: (C2 − C5)/G7; GαsS−CAG: (G7 − C2)/G7; GαsL+CAG: (C12/T9) × (C5/G7); GαsL−CAG: (T11/T9) × (C5/G7). CLL: blood mononuclear cells from patients with chronic lymphatic leukemia (n = 16), AT: adipose tissue (n = 26), UT, urothelial tumor tissue (n = 12), RB: human retinoblastoma cell lines (n = 7), LB: B lymphoblast cell lines (n = 17), HT: heart muscle tissue (n = 12), BR: brain tissue (n = 3).
Figure 7
Figure 7
Relative Gαs expression in isoproterenol-stimulated EBV-transformed B lymphoblasts. Lymphoblasts (5 × 106) from four different individuals were stimulated with isoproterenol (100 nM) and relative mRNA expression was determined at indicated time points using real-time PCR analysis. RNA (1 μg) was used for the RT reaction and all expression levels were measured in duplicate in the same assay. Relative expression levels are given as Ct values of the maximum of cycles (45) minus measured Ct values. No significant changes in Gαs expression were detectable. Fold change of expression at 8 and 24 h compared with baseline (0 h) using the 2−ΔCt formula was 0.9 and 1.04. Data are expressed as means ± SEM.
Figure 8
Figure 8
Isoproterenol-induced changes of Gαs splice variant ratios. Lymphoblasts (5 × 106) from four different individuals were stimulated with isoproterenol (100 nM) and splice variant ratios were determined at indicated time points as described in Materials and Methods. Isoproterenol stimulation resulted in a slight decrease in GαsS/Gαs ratio with a maximum after 8 h (A) and a corresponding increase in GαsL/Gαs ratio (B). Within the short variant, the ratios of +/−CAG variants changed significantly after 4 h of isoproterenol treatment with an increase of +CAG variants and a decrease of −CAG variants. The GαsS+CAG/GαsS ratio increased from 0.55 ± 0.02 at baseline to a maximum of 0.64 ± 0.02 after 4 h (p < 0.05). After 24 h, baseline values were reached again (C). The same effect was observed for +/−CAG variants within the long variant. The GαsL+CAG/GαsL ratio increased from 0.08 ± 0.01 at baseline to a maximum of 0.17 ± 0.01 after 4 h (p < 0.05) and returned to baseline values after 24 h (D). All experiments were repeated at least twice at different time points with similar results. Data are expressed as means ± SEM.
Figure 9
Figure 9
Comparison of Pyrosequencing and real-time PCR method for quantifying Gαs splice variants. Three retinoblastoma and three CLL cDNAs were analyzed for Gαs total to Gαs long ratios. The same cDNAs were used for comparing Pyrosequencing and real-time PCR results. Pyrosequencing results in 5.36 ± 0.093 ratio for CLL and 2.49 ± 0.158 for retinoblastoma (p < 0.0001), whereas real-time PCR using SYBR green leads to 4.79 ± 0.382 and 2.51 ± 0.326 (p < 0.05), indicating Pyrosequencing to be the more precise method for quantification of splice variants.
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