Structural analysis of transcripts for the protein L-isoaspartyl methyltransferase reveals multiple transcription initiation sites and a distinct pattern of expression in mouse testis: identification of a 5'-flanking sequence with promoter activity.

The gene encoding the protein L-isoaspartyl-(D-aspartyl) methyltransferase (protein carboxyl methyltransferase, PCMT) is widely expressed in bacteria and eucaryotic cells. An antisense probe encompassing the first exon of the murine PCMT gene [E. A. Romanik, C. L. Ladino, S. C. D'Ardenne, and C. M. O'Connor (1992) Gene 118, 217-222] was used in ribonuclease protection assays to identify the initiation sites for PCMT transcription in mouse testis, brain, and liver tissues. Two major initiation sites, 155-157 nucleotides (nt) and 119 nt upstream from the ATG initiation codon, were identified in all tissues in addition to several minor sites. The locations of the initiation sites in testicular RNA were confirmed using ligation-mediated 5'-rapid amplification of cDNA ends (RACE). These initiation sites are situated at the 3'-end of a 407-bp genomic sequence which is sufficient to drive the expression of a firefly luciferase gene in transient transfection assays with NIH/3T3 cells. The 407-bp sequence resembles a housekeeping gene promoter in its high G+C content, lack of a TATA box and the presence of multiple potential binding sites for the transcription factors Sp1 and ETF. Alternative splicing in the C-terminal encoding sequence and in the 3'-untranslated regions of PCMT transcripts generates three distinct classes of mRNAs which were cloned from testicular poly(A)+ RNA using 3'-RACE. Transcript splicing either 38 nt downstream or 7 nt upstream from the termination codon in exon 7 produces mRNAs encoding PCMT isozymes with -RWK or -RDEL, respectively, at their C-termini. The predominant transcript in testis, which is not detected in somatic tissues by Northern blotting and which may be specific to germ cells, is not spliced within exon 7 and also encodes the -RWK isozyme.