Human Melanocytes and Melanomas Express Novel mRNA Isoforms of the Tyrosinase-Related Protein-2/DOPAchrome Tautomerase Gene: Molecular and Functional Characterization

Abstract

We previously reported that a melanoma antigen, recognized by tumor-specific cytotoxic T lymphocytes, was encoded by intron sequences retained in a partially spliced transcript of the tyrosinase-related protein-2/DOPAchrome tautomerase gene. At difference with the mRNA encoding tyrosinase-related protein-2, this anomalous transcript was not expressed in melanocytes. This study examined whether neoplastic and/or normal cells of the melanocytic lineage could express additional forms of tyrosinase-related protein-2 mRNA. Screening of a melanoma-derived cDNA library with a tyrosinase-related protein-2 probe allowed identification of two novel isoforms. The first, tyrosinase-related protein-2-long tail, corresponds to the dominant transcript detected on melanomas and melanocytes by northern blot analysis. Tyrosinase-related protein-2-long tail is identical to the tyrosinase-related protein-2-encoding published cDNA sequence except for an extended 3'-untranslated region and is originated by alternative polyadenylation. This novel 3'-untranslated region contains an alternatively spliced, tyrosinase-related protein-2 last exon in the second isoform (tyrosinase-related protein-2-8b). The protein encoded by tyrosinase-related protein-2-8b is identical to tyrosinase-related protein-2 in its first 460 amino acids but possesses a different carboxyl-terminus devoid of transmembrane domain. Tyrosinase-related protein-2-long tail exhibited DOPA-chrome tautomerase activity, when transiently transfected into COS-7 cells. On the contrary, no detectable activity was exhibited by tyrosinase-related protein-2-8b. Reverse transcription-polymerase chain reaction analysis indicated that tyrosinase-related protein-2-long tail and tyrosinase-related protein-2-8b are expressed by tyrosinase-related protein-2-positive melanomas and normal melanocytes. Moreover all cell lines positive for tyrosinase-related protein-2 isoforms expressed tyrosinase and, all but one, tyrosinase-related protein-1. These data show that the human tyrosinase-related protein-2/DOPAchrome tautomerase gene can yield different isoforms by alternative poly(A) site usage or by alternative splicing. The pattern of expression of these isoforms suggest that they might play a part in the normal pathway of melanin biosynthesis.

Pre-Existing Immunity to Tyrosinase-Related Protein (TRP)-2, a New TRP-2 Isoform, and the NY-ESO-1 Melanoma Antigen in a Patient with a Dramatic Response to Immunotherapy

“…In contrast, the isoform TRP-2-INT2, which contains an HLA-A68-restricted CTL epitope, encodes a shortened protein of 237 amino acids because of an early stop site located in the retained intron 2 (31). Two other isoforms, TRP-2-8b and TRP-2-LT, have been described but have not been shown to contain any novel CTL epitopes (30). TRP-2-8b contains a novel exon 8b but lacks exon 8 and therefore, like TRP-2-INT2, is devoid of transmembrane domain as well as the DOPAchrome tautomerase activity.…”

Extraction of Transcript Diversity from Scientific Literature

“…For example, work by Pisarra et al [36] on human Dopachrome tautomerase describes two transcripts in melanocytes and melanomas with a “different carboxyl-terminus” generated, concluding that “dopachrome tautomerase can yield different isoforms by alternative poly(A) site usage or by alternative splicing” (Figure 4). …”

“…Using the heaviest bundling algorithm [37] with genomic sequence data from Ensembl [38], and transcript data from UniGene [39] clusters for the gene, we were able to generate two transcript isoforms for Dopachrome tautomerase (Figure 4, bottom) resembling those described by Pisarra et al [36] and were able to detect an AS event in the 3′ region. Hence, the use of large-scale methods may provide detailed information about underlying events, and text mining would provide functional annotations to the transcript isoforms observed.…”

RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi

“…TRP-1 and TRP-2 are associated with distinct patterns of melanocyte distribution and function in normal skin and cutaneous pigmented lesions. 19,20 A recent study using quantitative PCR and cDNA array hybridization of tissues prepared by laser pressure catapulting verified high expression of TRP-2 by nevi and melanomas. 21 TRP-2 is involved in the protection of melanoma cells from apoptosis.…”