Mammalian glutaminase Gls2 gene encodes two functional alternative transcripts by a surrogate promoter usage mechanism

Abstract

Background: Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene.

Methodology/principal findings: We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed.

Conclusions/significance: This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals.

Figure 1. Structure of the human GLS2 gene and predicted GA transcripts.

The GLS2 gene is located in the 12q13 region of human chromosome 12, as shown in the upper part of this figure. The gene has a length of approximately 18 kb and split into 18 exons. Exon sequences are indicated as numbered light green boxes; intron sequences are shown as solid blue lines. Two GA transcripts are encoded by GLS2: the canonical GAB mRNA formed by joining the full 18 exons of the gene and the short LGA transcript that lacks exon 1. Dotted lines comprise exons involved in the generation of both transcript variants. The transcription start site is marked by an arrow and numbered as +1.

Figure 2. Comparison of the sequences of RT-PCR products demonstrating co-expression of LGA and GAB transcripts in mouse, rat and human tissues.

The identity of each amplified fragment was assessed by sequence alignment with known sequences for rat liver LGA (GenBank# J05499) and human GAB (GenBank #AF348119) using Blast program. Top panel: the 5′-sequences of mouse brain and human liver LGA cDNA fragments obtained by RT-PCR were aligned with the sequence of rat liver LGA cDNA; bottom panel: the 5′-sequences of rat liver and mouse brain GAB cDNA fragments obtained by RT-PCR were aligned with the human GAB sequence from ZR-75 breast cancer cells. Identical nucleotides are indicated in red, different nucleotides are labeled in blue. Sequence alignment was done using Multalin program (http://multalin.toulouse.inra.fr/multalin/).

Figure 3. Nucleotide and deduced amino acid sequences of human LGA transcript.

The short LGA transcript variant was cloned by RT-PCR from human brain mRNA as described in the Materials and Methods section. The cloned cDNA was fully sequenced: nucleotide and deduced amino acid sequences are shown. The two initials ATG start codons are labeled in red; 5′-UTR and 3′-UTR regions are marked in yellow. Amino acid 1 is the initiation methionine and the stop codon is indicated by the asterisk. Sequences were arranged using Prettyseq program (http://emboss.sourceforge.net/apps/release/6.4/emboss/apps/prettyseq.html).

Figure 4. Primer extension analysis of human brain RNA.

Oligonucleotides GSP-6, PEXT1 and PEXT2 flanking the 5′ termini of the human LGA cDNA clone were ³²P end-labeled with [γ-³²P]ATP and T4 polynucleotide kinase. Poly(A)⁺ mRNA of human brain (approx. 0.5–1 µg) was mixed with the radioactive oligonucleotides and reverse-transcribed with avian myeloblastosis virus reverse transcriptase; the products from primer extension reactions were separated by electrophoresis on an 8% (w/v) denaturing polyacrylamide gel. Lanes are labeled with the name of the specific primer used in each case. The size of the single-stranded cDNA products (in bases) was estimated approximately by comparison with ФX174 HinfI DNA markers shown at the left in lane M. C+ lane: product amplified with a positive control of kanamycin. C- lanes: negative controls using PEXT2 with mRNA omitted. The black arrow indicates the specific GA products of approx. 140 bases obtained by reverse transcription with primer PEXT2. Top panel: scheme showing the approximate position of the three primers with regard to the TSS (+1) and the two first ATG codons of human LGA transcript.

Figure 5. Main transcription factor recognition sites presented in the alternative promoter of the human LGA transcript.

Schematic diagram showing the 7,341 kb of intron 1 of the human GLS2 gene. The transcription start site is indicated by +1. The sequences from nt -7340 to nt -6540 (enhancer distal region) and from nt -1500 to nt +1 (proximal promoter) are represented. Main putative protein binding sites, determined by computer analysis, are identified by vertical bars and are named in bold letters below the sequence. The program TESS (http://www.cbil.upenn.edu/tess) was employed for searching consensus transcription factor motifs. A second p53 motif, found in between distal and proximal promoters, is shown in normal type characters.

Figure 6. ENCODE Enhancer- and Promoter-Associated Histone Marks, CpG islands and methylation status of the GLS2 gene locus on human chromosome 12.

For simplicity, only the four first exons of human GLS2 gene and the 5′-flanking genomic region are shown on top of the Figure. Below the GLS2 gene, six plots are represented, as follows: first panel: Enhancer- and Promoter-Associated Histone mark (H3K4Me1) from 9 human cell lines; second panel: Promoter-Associated Histone mark (H3K4Me3) from 8 human cell lines; third panel: Enhancer- and Promoter-Associated Histone mark (H3K27Ac) from 8 human cell lines; fourth panel: CpG island shown as a solid green bar; fifth panel: Promoter-Associated Histone mark (H3K4Me3) from chromatin immunoprecipitation of DNA and sequencing data (ChIP-seq) and; sixth panel: Methylation-dependent immunoprecipitation of DNA and sequencing data from human brain (MeDIP-seq).

Figure 7. Quantification of mRNA levels of KGA, GAB and LGA transcripts in rat and mouse tissues by qPCR.

The histogram shows the absolute copy number of mRNA transcripts for the KGA (dark blue), GAB (pink) and LGA (light blue) isoforms. Real time RT-PCR was performed as indicated in the Materials and Methods section. Results are mean ± S.E.M. of three independent experiments done in triplicate. Values were determined for liver and brain tissues from rat and mouse and are shown as copy number per ng of total RNA. Insets: GAB and LGA mRNA values in mouse and rat brains are depicted without KGA mRNA levels to appreciate differences among them.

Figure 8. In vitro transcription and translation of human LGA cDNA.

The ORF of human LGA (hLGA), deletion mutant hLGA (Mut-hLGA) starting at the second ATG, and Δ4- and Δ10-LGA deletion mutants were cloned into the pGEM-T vector and transcribed and translated in vitro in the presence of ³⁵S-Met. The reaction mixtures were then analyzed by SDS-PAGE and autoradiography. Lane M, standard protein markers with the relative positions of prestained molecular mass markers indicated on the left; lane Mut-hLGA, aliquot of the translation mixture using the human LGA cDNA starting at the second in-frame ATG; lane hLGA, full-length human LGA; lane Mut-Δ4, aliquot of the translation mixture using the Δ4-LGA deletion mutant cDNA; lane Mut-Δ10, aliquot of the translation mixture using the Δ10-LGA deletion mutant cDNA; lane C-, negative control (pGEM-T with the hLGA cloned in the antisense orientation); lane C+, luciferase positive control. The radioactive LGA protein bands are indicated on the left using black arrows.

Figure 9. Glutaminase activity assay.

The in vitro transcribed and translated human LGA (hLGA) protein and a deletion mutant (Mut hLGA) protein, starting at the second ATG initiation codon, were assayed for GA activity as described in the Materials and Methods section. Results are mean ± S.E.M. of three independent experiments: protein hLGA (pink bar); protein Mut hLGA (blue bar). Background activity was measured by using the pGEM-T[hLGA] construct cloned in the antisense orientation and was always subtracted from activity values obtained with the hLGA proteins. Values are expressed as milliunits of enzyme activity per ml.

Figure 10. Immunoblot analysis of human SHSY-5Y neuroblastoma cells and rat liver and brain mitochondria.

Cell extracts of human neuroblastoma SHSY-5Y cells (left and center panel) and mitochondria isolated from rat liver and brain (right panel) were analyzed by SDS-PAGE and Western blotting. Blots were revealed by chemiluminescence using polyclonal antibodies raised against the exclusive first exon of human GAB protein (left panel) or against the whole human GAB protein which recognize both GAB and LGA proteins (center and right panel). M, lanes containing the molecular mass markers indicated on the left margin; SHSY-5Y, lanes loaded with protein extracts isolated from human neuroblastoma cells; Liver 1 and 2: lanes containing total liver protein extracts and mitochondrial protein extracts, respectively; Brain 3 and 4: lanes containing total brain protein extracts and mitochondrial protein extracts, respectively.