The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Abstract

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.

Figure 1

Structural map of the XPG gene. The 15 exons and 14 introns are numbered and their size in base pairs is indicated below. The parts of the genomic XPG sequence that we determined are indicated and were submitted to GenBank (accession nos AF255431–AF255442). AL137246 and AL157769 are unannotated sequences that were subsequently found in GenBank.

Figure 2

Structural map of alternatively spliced XPG mRNA isoforms. To identify the isoforms, total RNA was reverse transcribed, RT–PCR amplified and subsequently subcloned into a cloning vector for sequencing (see Materials and Methods). The splice isoforms (I–VI, bottom) are compared to the wild-type XPG mRNA (middle) and the wild-type genomic XPG DNA (top). The genomic location and size (in base pairs) of insertions/deletions due to alternative splicing are indicated.

Figure 3

XPG mRNA: normal and variant splice isoforms in human tissues. Top, cultured normal primary skin fibroblasts (F1-AG05247E and F2-AG05410). RNA was isolated from each cell line, cDNA was prepared by RT–PCR and amplified separately by use of primer pairs that cover both normal splice isoforms and each of the variant splice isoforms (I–VI). The PCR products were separated by agarose gel electrophoresis (see Materials and Methods). Each pair of lanes present results from F1 and F2, respectively, for each of the primer pairs. The black arrows indicate the location of the normally spliced isoforms. Detectable alternatively spliced isoforms are indicated by white arrows. The asterisk indicates a heterodimer of the normal and the abnormally spliced isoforms (II and VI). The normal isoform is present in each lane. Alternatively spliced isoforms II and VI are seen on the gel indicating their relative abundance. Middle and bottom, cDNA obtained from human brain, liver, lung, kidney, spleen and prostate tissue was amplified by use of primer pairs for XPG mRNA isoform II (middle, left), isoform IV (middle, right) and isoform VI (bottom) and separated by agarose gel electrophoresis. The black arrows indicate the normally spliced isoforms. The alternatively spliced isoforms (white arrows) and heterodimers between the normal and alternatively spliced forms (asterisk) are detectable in the different tissues. There appears to be a relative reduction in alternatively spliced isoforms II, IV and VI in the kidney compared to the other human organs.