Novel isoform of the Xenopus tropicalis PKA catalytic alpha subunit: An example of alternative splicing

Abstract

The cAMP-dependent protein kinase (PKA) plays key roles in the control of various aspects of eukaryotic cellular activities by phosphorylating several proteins and is multifunctional in nature. In the case of frog, Xenopus tropicalis, a gene encoding the PKA catalytic alpha subunit has been identified which encodes a single protein. Here we report the occurrence of N-terminal alternative splicing events in X. tropicalis tadpole that, in addition to generating a myristoylatable isoforms, also generate the non-myristoylated variant of the catalytic alpha subunit as has been reported in various other organisms. In addition to the already characterized exon 1, the 5' untranslated region and first intron actually contains one more other exon, that is alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with already characterized all internal exons. Thus, X. tropicalis tadpole expresses at least two different isoforms of the catalytic alpha subunit of PKA. The significance of this structural diversity in the family of PKA catalytic subunits is discussed.

Figure 1. Schematic diagramme and exon/intron organization of the gene encoding PKA C-subunit of X. tropicalis

(A) Schematic presentation of exon-intron organization showing exons with filled boxes and introns with a thick line joining all the exons. Expressed exons and their splicing pattern are shown by thin lines as reported earlier (Klein et al., 2002). Exons/Introns are not drawn to the scale.

Figure 2. 5′ RACE of X. tropicalis mRNA

5′ Rapid Amplification of cDNA Ends (5′ RACE) was independently performed three times using PKA C-subunit encoding gene specific internal primer and RNA isolated from mixed tadpoles of X. tropicalis. M indicates DNA size marker, +RT indicates reverse transcriptase was added and −RT indicates no reverse transcriptase was added to the reaction mixture.

Figure 3. RT-PCR analysis of predicted spliced variant of the PKA C-subunit gene of X. tropicalis

(A) Splicing pattern of newly identified cDNA having new exon encoded by first intron of the gene replacing the first reported exon. Exons reported earlier are shown with filled boxes and introns with a thick line joining all the exons. The newly identified exon is shown with a rectangle with vertical lines insisde. Exons/Introns are not drawn to the scale. (B) RT-PCR amplification was used to determine the presence of transcripts, containing predicted exon, in total RNA prepared from mixed-stage X. tropicalis tadpoles as described in the Materials and methods section. This experiment was independently performed three times. M indicates size marker on the left. RT-PCR products were obtained using a common reverse primer and exon specific forward primers representing each spliced variants. Lane 1 represents RT-PCR product of the reported gene product and Lane 3 represents newly identified gene products. Lanes 2 & 4 represents the seminested PCR of products obtained from lanes 1 & 3 respectively when common reverse primer was changed to another primer internal to the first primer. Anticipated sizes of the products are 840, 459,940 and 559 bp in lanes1-4 respectively.

Figure 4. Multiple sequence alignment (CLUSTALW) of published and new isoform of PKA catalytic alpha subunit of X. tropicalis

Multiple sequence alignment of alternatively spliced two isoforms of PKA C-subunit of X. tropicalis. The amino acid sequences of earlier reported myristoylatable isoform (as published) and newly identified non-myristoylatable isoform (as New) were aligned using CLUSTALW at www.ebi.ac.uk/clustalw. Asterisks indicate the identical residues and rest are either similar (.) or non-identical. Arrow indicated above the sequence is the junction of exon-1 and exon-2. Continuous stretch of asterisks under the sequence represents the common sequence present in both the isoforms starting from exon-2. Nucleotide sequence of the new transcript and encoded amino acid sequence of was submitted to EMBL database and the accession number FN562397 is mentioned at the end of the sequence.

Figure 5. Semi-quantitative RT-PCR for relative abundance of both the transcripts

(A) Total RNA isolated from tadpole was reverse transcribed using a gene specific reverse primer corresponding to exon-4. Product thus obtained was equally distributed and PCR was performed using forward primer. M indicates DNA size marker on the left and right of the gel. Lanes 1-5 present RT-PCR products obtained using forward primer from earlier reported first exon for 22, 24, 26, 28 and 30 cycles. Lanes 6-10 present RT-PCR products obtained using forward primer from newly identified exon for 22, 24, 26, 28 and 30 cycles. (B) A histogram showing the relative intensities of the DNA bands of figure 5 (A). The experiment was repeated five times independently and the average was taken for the histogram presentation. Data represent means ± respective standard errors. Bars differ significantly (P ≤ 0.05).