Human melanocytes and melanomas express novel mRNA isoforms of the tyrosinase-related protein-2/DOPAchrome tautomerase gene: molecular and functional characterization.
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.
Pisarra
P
P
Human Tumors Immunobiology, Department of Experimental Oncology, National Cancer Institute, Milan, Italy.
Lupetti
R
R
Palumbo
A
A
Napolitano
A
A
Prota
G
G
Parmiani
G
G
Anichini
A
A
Sensi
M
M
D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D008544:Melanocytes / Q000737:chemistry*; D008545:Melanoma / Q000235:genetics*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000302:isolation & purification / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D020033:Protein Isoforms; D012333:RNA, Messenger; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase