Characterization of DNA polymerase beta splicing variants in gastric cancer: the most frequent exon 2-deleted isoform is a non-coding RNA

Abstract

DNA repair polymerase beta (Pol beta) gene variants are frequently associated with tumor tissues. In this study a search for Pol beta mutants and splice variants was conducted in matched normal and tumor gastric tissues and blood samples from healthy donors. No tumor associated mutations were found while a variety of alternative Pol beta splicing variants were detected with high frequency in all the specimens analysed. Quantitative PCR of the Pol beta variant lacking exon 2 (Ex2Delta) and the isoforms with exon 11 skipping allowed to clarify that these variants are not tumor- neither tissue-specific and their levels vary greatly among different individuals. The most frequent Ex2Delta variant was further characterized. We clearly demonstrated that this variant does not encode protein, as detected by both western blotting and immunofluorescence analysis of human AGS cells expressing HA-tagged Ex2Delta. The lack of translation was confirmed by comparing the DNA gap-filling capacity and alkylation sensitivity of wild type and Pol beta null murine fibroblasts expressing the human Ex2Delta variant. We showed that the Ex2Delta transcript is polyadenylated and its half-life is significantly longer than that of the wild type mRNA as inferred by treating AGS cells with actinomycin D. Moreover, we found that it localizes to polyribosomes suggesting a role as post-transcriptional regulator. This study identifies a new type of DNA repair variants that do not give rise to functional proteins but to non-coding RNAs that could either modulate target mRNAs or represent unproductive splicing events.

Fig. 1

Pol β splicing variants in human gastric tissues and gastric cell lines (A) Analysis of Pol β cDNA by RT-PCR of mRNA from gastric tissues or cell lines. Primers were designed to amplify the entire Pol β coding sequence. Lane 1: DNA marker; lane 2: normal (N) and lane 3: tumor (T) tissues of one GC patient (code 88); lane 4: AGS gastric cancer cell line. (B) Nested-PCR of amplified Pol β cDNA from normal (N) and tumor (T) tissue of one GC patient (code 17). The 200 bp fragment represents the wild type amplicon. Lower molecular weight products indicate the occurrence of splicing events. The skipping of exon 2 (lane 1–2), exon 5+6 (lane 3–4) and exon 9 (lane 5–6) is shown.

Fig. 2

QPCR analysis of Pol β wild type and splicing variant mRNAs in normal and tumor tissues. Expression levels were measured in paired normal and tumor gastric specimens from 7 gastric cancer patients and gastric and blood samples from healthy individuals. (A) Pol β wild type, (B) Ex2Δ and (C) Ex11Δ isoforms mRNA levels in tumor tissues relative to paired normal tissues. (D) Ex2Δ Pol β transcript levels in normal and tumor tissues relative to wild type Pol β mRNA levels in the same tissues. Gastric cancer patients are indicated by code. Specimens from healthy subjects are indicated as follows: gastric mucosa (N1 and N2) and blood samples (B1 to B4). The mean +/− SD of three independent experiments is shown.

Fig. 3

Western blot and immunofluorescence analysis of Pol β in mouse fibroblasts and human AGS cells. (A) Wild type and Pol β defective mouse fibroblasts were stably transfected with wt and Ex2Δ Pol β cDNA. Lane 1: Full length and 8-kDa domain Pol β recombinant proteins. Lane 2: Pol β wt cells. Lane 3: Pol β null cells. Lane 4: Pol β null cells transfected with human wild type Pol β. Lane 5: Pol β null cells transfected with the Ex2Δ Pol β variant. Lane 6: Pol β wt cells transfected with the Ex2Δ Pol β variant. (B) AGS cells were transiently transfected with HA-tagged wt and Ex2Δ Pol β cDNA. Lane 1: AGS cells transfected with HA-Ex2Δ Pol β. Lane 2: AGS cells transfected with HA-Ex2Δ Pol β and incubated with MG132. Lane 3: AGS cells transfected with HA-wt Pol β. Lane 4: AGS cells transfected with HA-wt Pol β and incubated with MG132. Lane 6: AGS cells untransfected. All the lanes framed in the same box belong to the same autoradiography although some lanes between samples were cut because not relevant. (C) AGS cells transfected with HA-wt Pol β (upper panel) and incubated with MG132 (lower panel). (D) AGS cells transfected with HA-Ex2Δ Pol β (upper panel) and incubated with MG132 (lower panel).

Fig. 4

Characterization of Pol β mouse cells transfected with the Ex2Δ Pol β variant. Cell growth rate after MMS treatment of (A) Pol β defective cells: Pol β null (), Pol β null/wt (), Pol β null/Ex2Δ () and (B) Pol β wild-type cells : Pol β wt () and Pol β wt/Ex2Δ () cells. Bars represent standard deviation. (C) Gap-filling assay performed with cell extracts from Pol β wt (lanes 1–3), Pol β null (lanes 4–6), Pol β null/wt (lanes 7–9), Pol β null/Ex2Δ (lanes 10–12) and Pol β wt/Ex2Δ cells (lanes 13–15). A typical experiment is shown.

Fig. 5

(A) Analysis of Pol β splicing variant polyadenylation. RT-PCR was performed on total RNA (1µg) from AGS gastric cancer cell line by using random examers (lane 1) or oligo dT (lane 2). Primers which recognize the entire coding sequence of Pol β cDNA were used in a second PCR to obtain Pol β splicing variants amplification. The position in the gel of the 1353 bp marker is shown. Multiple bands represent Pol β splicing variants. (B) Analysis of wild type and Ex2Δ Pol β mRNA stability performed by QPCR on RNA of AGS gastric cancer cell line treated with actinomycin D. The mean +/− SD of four independent experiments is shown. (C) Polyribosome distribution of wild type (–) and Ex2Δ (---) Pol β mRNA. Human 18S (– –) was used as positive control. The contents of each transcript were quantified by QPCR. The fraction with the highest level of transcript was used as reference (=1).