Characterization of the Mcoln1 alternative splice variant
In order to determine the coding sequence for the larger transcript, we searched the mouse EST database using each intron as well as the genomic sequence flanking the Mcoln1 gene. Two ESTs were identified that contained sequence from intron 12 (GB No. AI430291 and AA874645), and the corresponding clones were sequenced. Clone 408619 (ESTs: GB No. AI430291, AI429558) begins approximately 1.1 kb before exon 13 and continues through the exon and splices correctly to exon 14. Clone 1281641 (EST:GB No. AA874645) begins 175 bp before exon 13 and also splices correctly to exon 14. A mouse multiple tissue Northern was hybridized using a probe generated from the putative intron sequence in clone 408619 (Probe 2, Fig. 1B), which detected only the 4.4 kb band (Fig. 3C).
In order to determine the sequence of the entire transcript, RT-PCR using primers in exons 10 and 11 paired with a primer in intron 12 was performed using BALB/c mouse brain total RNA and the resulting products sequenced. These products show that the larger transcript is due to an alternative splice event that results in an expanded exon 13. Specifically, exon 12 splices at bp 436 of intron 12, creating a large 1614 bp exon 13 that splices correctly to exon 14. The open reading frame of this alternatively spliced transcript is 611 amino acids, 28 amino acids longer than the message encoded by the 2.4 kb transcript.
TMPred analysis predicts that isoform 2 encodes a protein identical in structure to Mcoln1, possessing 6 transmembrane domains and a channel pore, however the protein sequences diverge at amino acid 526. The 55 amino acid C-terminal cytoplasmic tail encoded by the 2.4 kb transcript is completely different from the 86 amino acid tail encoded by the murine specific 4.4 kb transcript (Fig. 4). Clontech Mouse RNA Master Blots were hybridized with the exon 2 and intron 12 probes mentioned above in an attempt to determine if these two transcripts showed differences in expression patterns, however, there was no significant difference in the 22 tissues represented (data not shown).
Figure 4  Peptide sequence comparison of the two alternatively spliced Mcoln1 isoforms. The green box surrounds the divergent c-terminal cytoplasmic tails. The blue lines indicate the transmembrane domains. Next, we directly compared the nucleotide and amino acid sequence of the alternatively spliced mouse transcript to the entire human MCOLN1 genomic sequence and found no significant similarity. As mentioned previously, Northern blots performed with human MCOLN1 probes show only one 2.4 kb transcript. In addition, we hybridized a human multiple tissue Northern and human Southern with a probe in human intron 12 that is adjacent to exon 13. The probe was located in the region syntenic to that which encodes the alternate mouse transcript. Only the expected bands were detected on the Southern and no bands were detected on the Northern, confirming that this alternative transcript is specific to murine Mcoln1. Recent BLASTP analysis of the alternate Mcoln1 transcript yields a match to a putative 145 amino acid anonymous protein (GB No. BAB25862) predicted from a RIKEN clone. It is obvious from our results, however, that the identification of this sequence as a full-length protein is incorrect since probes unique to the clone, as well as probes containing the Mcoln1 coding sequence, identify the same transcripts.