Exclusion of exon 2 is a common mRNA splice variant of primate telomerase reverse transcriptases

Abstract

Telomeric sequences are added by an enzyme called telomerase that is made of two components: a catalytic protein called telomerase reverse transcriptase (TERT) and an integral RNA template (TR). Telomerase expression is tightly regulated at each step of gene expression, including alternative splicing of TERT mRNA. While over a dozen different alternative splicing events have been reported for human TERT mRNA, these were all in the 3' half of the coding region. We were interested in examining splicing of the 5' half of hTERT mRNA, especially since exon 2 is unusually large (1.3 kb). Internal mammalian exons are usually short, typically only 50 to 300 nucleotides, and most long internal exons are alternatively processed. We used quantitative RT-PCR and high-throughput sequencing data to examine the variety and quantity of mRNA species generated from the hTERT locus. We determined that there are approximately 20-40 molecules of hTERT mRNA per cell in the A431 human cell line. In addition, we describe an abundant, alternatively-spliced mRNA variant that excludes TERT exon 2 and was seen in other primates. This variant causes a frameshift and results in translation termination in exon 3, generating a 12 kDa polypeptide.

Figure 1. Molecular architecture of the hTERT coding region.

Known hTERT protein domains and motifs are shown. The 1132-amino acid protein is aligned above the 3396-nt coding region of the 16 hTERT exons. Common alternatively spliced variants are shown below the wild type mRNA. These include the α-variant (deletion of first 36 nucleotides from exon 6), the ß-variant (deletion of exon 7 and 8) and the DEL[e2] variant (deletion of exon 2) described herein. The predicted open reading frame (ORF) for each mRNA is indicated. The 5′ UTR and 3′ UTR are not shown.

Figure 2. Deletion of exon 2 is a common alternative splice variant of hTERT mRNA.

Amplified hTERT exon-exon junctions in an RT-PCR reaction of total RNA from A431 cells. hTERT exons, the location of each primer pair and the amplicon sizes predicted from wild type hTERT cDNA are indicated. The fraction of PCR products from alternatively spliced variants is shown below the gel. Wild type amplicons are represented by arrowheads, abundant alternatively spliced variants are indicated by an asterisk. Non-specific bands are indicated as “a” (actin bundling protein) and “b” (CDC27).

Figure 3. A competitive deletion hTERT construct results in amplicons that are approximately 10% smaller than wild type.

A. Competitive PCR cDNA construct was generated by deleting 5 regions (D1–D5, black boxes) from wild type hTERT cDNA plasmid. Approximately 10% of each amplicon was deleted. B. PCR amplicons of plasmid DNA from wild type hTERT (W) and the competitive deletion hTERT construct (D) resolved on a 2% agarose gel.

Figure 4. There are approximately 20–40 molecules of hTERT mRNA per cell.

A. The in vitro transcribed deletion hTERT RNA was added to total cellular mRNA from A431 cells in varying amounts to generate a titration curve between 1×10⁴ and 1×10⁷ molecules of competitive RNA. This RNA sample was reverse transcribed so that both competitor and endogenous hTERT could be amplified in the same PCR reaction to determine the initial amount of hTERT mRNA present in the reaction. B. The forward primer was radiolabeled and hTERT RT-PCR of total A431 RNA was performed in the presence of hTERT competitive deletion constructs. Samples were separated on a 4% denaturing acrylamide sequencing gel. The identity of each band is indicated by the exon structure to the right. The amplicon is indicated by the dotted line above the diagrams. Data are representative of at least two independent experiments. DEL, deletion; INS, insertion; e, exon; i, intron.

Figure 5. hTERT exon 2 is underrepresented relative to the flanking exons in deep sequencing datasets.

The percent inclusion of hTERT exon 2, relative to either exon 1 or exon 3 (whichever was higher) was determined from 78 deep sequencing datasets. Datasets were binned into inclusion rates of 0–25%, 26–50%, 51–75% and 76–100%. TERT from ES cells contained high levels of exon 2, in contrast to other cell lines and primary tissues.

Figure 6. Large internal exon 2 is conserved in mammals.

Genomic nucleotide sequences from the end of exon 1 to the beginning of intron 2 for human, chimpanzee, gorilla, macaque, cow, pig, dog, elephant, mouse and rat were aligned using Jalview. The locations of the human protein domains encoded by these nucleotide sequences are indicated above. Nucleotide residues are colored according to the percentage of residues in each column that are identical to the human sequence (>80% mid blue, >60% light blue, >40 light grey, <40% white). Only those residues that match the human sequence are colored. The percent conservation at each nucleotide position across all of the species listed is also indicated in the bar graph in black at the bottom. Conservation is calculated according to the Jalview and AMAS methods. Exons, and specifically the protein motifs and domains, are more conserved than flanking regions. 20 nucleotides flanking the exon junctions are shown in detail. The splice site consensus sequences are indicated above each highlighted region and the exon-exon boundaries are marked with arrows.

Figure 7. Exclusion of exon 2 from hTERT mRNA is specific to primates.

A. A phylogenetic tree of TERT mRNA coding regions was created with the Jalview algorithm using percent identity neighbor joining. A genome sequence is currently unavailable for Bonobo, but is in the same genus (Pan) as chimpanzee. B. RT-PCR of TERT exons 1–3 of Monkey (Cos-7 cells), Mouse (NIH3T3), Rat (NRK), Dog (D17), Orangutan (Pongo pygmaeus) and Bonobo (Pan paniscus). An arrow indicates wild type bands and an asterisk indicates DEL[e2]. PCR reactions shown are representative of at least 2 trials.

Figure 8. In vitro translation of hTERT mRNA lacking exon 2.

Radiolabled in vitro transcription and translation of wildtype and DEL[e2] constructs in rabbit reticulocyte lysate. Wild type TERT produced the expected 122 kDa product, but a DEL[e2] product was not detected. Thus, sequences encoding 30 kDa of Upf3A were inserted into both constructs immediately after the hTERT start codon in exon 1. Protein products for wildtype (152 kDa) and DEL[e2] (42 kDa) were then detected. Products are separated by 4–20% gradient SDS-PAGE.