In Silico characterization of phosphorylase kinase: evidence for an alternate intronic polyadenylation site in PHKG1

Abstract

Phosphorylase kinase (PhK), the key enzyme that regulates glycogenolysis, has traditionally been thought to be expressed predominantly in muscle and liver. In this study, we show by two different database searches (Expressed Sequence Tag and UniGene) that PhK gene expression occurs in at least 28-36 different tissues, and that the genes encoding the alpha, beta, and gamma subunits of PhK undergo extensive transcriptional processing. In particular, we have identified exon 6 of PHKG1 as a 3' composite terminal exon due to the presence of a weak polyadenylation and cleavage site in intron 6. We have verified biochemically that transcriptional processing of PHKG1 does occur in vivo; mRNA corresponding to the alternate variant is expressed in skeletal muscle, brain, heart, and tongue. In silico translation of this mRNA yields a PhK gamma subunit that contains the first 181 residues of the protein, followed by an additional 21 amino acids. The implication of this alternate processing is discussed within the context of gamma catalysis and regulation.

Figure 1. Genomic Alignment of EST Sequences

The genomic location for each identified PHK EST sequence was determined as indicated in Materials and Methods. For each of the PHK genes shown (A–E), vertical lines and boxes represent exon regions while horizontal lines represent connecting intron regions. The tissue of origin for each identified EST is indicated on the right. Overlapping ESTs that were identified to encode for PHK exon regions were mapped to their genomic location by Sim4 analysis and are schematically represented here. The total number of nucleotides in each gene are as follows: PHKA1 – 133410 bp and Human Genome Locus (HGL) = NC_000023.9; PHKA2 – 91035 bp and HGL = NC_000023; PHKB – 237017 bp and HGL = NC_000016.8; PHKG1 – 12015 bp and HGL = NC_000007.12; PHKG2 – 8946 bp and HGL = NC_000016.8. Identified EST sequences are compared to the non-redundant RefSeq, the sequence that provides the intron/exon boundaries for each gene, and differences between ESTs and the RefSeq in a particular tissue represent alternative mRNA processing (underlined in red). Other highlighted regions include: exons 27–28 in PHKA1 previously reported to be alternatively spliced – blue circle; alternative splicing of PHKB in GenBank –purple circle; 3′ region of PHKG1 missing due to alternate polyadenylation - green. Two separate diagrams for PHKG2 expression in lung, placenta and brain are shown in order to emphasize the striking differential expression in the 5′ domain of this gene.

Figure 2. Species Conservation of the PHKG1 Polyadenylation and Cleavage Site

The DNA sequence of PHKG1 intron 6 is shown for six different species. The conserved hexanucleotide sequence is underlined; putative cleavage sites are indicated by arrows.

Figure 3. PCR amplification of the full-length and spliced PHKG1 variants

Human cDNA from brain, heart, small intestine, skeletal muscle, and tongue was used as the template for PCR as indicated in Materials and Methods. A. Amplification of full-length PHKG1 cDNA (1256 bp). B. Amplification of the 3′ truncated PHKG1 cDNA (795 bp). C. In silico translation of PHKG1 exon 6 with the additional 21 residues that result from alternate polyadenylation of intron 6 (underlined).