Abstract
Multiple AE2 Cl-/HCO3- exchanger mRNAs have been identified in rat. To determine the genetic basis for these mRNAs and whether they encode different variants of the exchanger, we used both rapid amplification of cDNA ends and S1 nuclease protection protocols and examined the organization of the gene. mRNAs encoding three N-terminal variants of AE2 (AE2a, AE2b, and AE2c) were identified and shown to be transcribed from alternative promoters. The AE2a transcription unit consists of 23 exons, with exons 1 and 2 containing 5'-untranslated sequence and the first 17 codons. The first exon of AE2b is located in intron 2; it contains 5'-untranslated sequence and an alternative 3-amino acid N-terminal coding sequence and is spliced to exon 3. The first exon of AE2c is located in intron 5; it consists of 5'-untranslated sequence and is spliced to exon 6, which contains the translation initiation codon corresponding to Met-200 of AE2a. Northern analysis shows that AE2a is expressed in all tissues, AE2b exhibits a more restricted distribution with highest levels in stomach, and AE2c is expressed only in stomach. Thus, the use of alternative promoters leads to the production of three N-terminal variants of AE2 that exhibit tissue-specific patterns of expression.
Highlights
Electroneutral ClϪ/HCO3Ϫ exchange in mammalian tissues is mediated by a family of proteins encoded by multiple mRNAs from at least three genes, termed AE1,1 AE2, and AE3
Cloning of the 5Ј Ends of mRNAs Encoding Three N-terminal Variants of AE2—Our previous Northern blot analyses [10] demonstrated the existence of at least three AE2 mRNAs. These include a 4.4-kb mRNA corresponding to the AE2 cDNA that has already been cloned, and mRNAs of 4.2 and 3.8 kb that seem to differ at their 5Ј ends
Use of Alternative Promoters Leads to Tissue-specific Expression of mRNAs Encoding N-terminal Variants of AE2—Earlier studies of the AE ClϪ/HCO3Ϫ exchanger gene family revealed the existence of multiple mRNAs for each of the three known genes [9, 10, 24, 25], and more recent analyses of the AE1 and AE3 genes have shown that some of these mRNAs are transcribed from alternative promoters and encode protein variants that differ in their N-terminal sequences [5,6,7,8, 11,12,13, 25,26,27]
Summary
Rapid Amplification of cDNA Ends (RACE) Protocol—The 5Ј RACE procedure was performed using the 5Ј-AmplifinderTM RACE Kit For amplification of the 5Ј end of AE2a described, the P1 primer was complementary to nt ϩ32 to ϩ52, and the P2 primer was complementary to nt ϩ17 to ϩ36 (numbered as in top panel of Fig. 7; see Fig. 5) In this experiment the P2 primer was 5Ј end-labeled with T4 kinase and [␥-32P]ATP before being used in the PCR reaction. Bacterial colonies harboring plasmids with AE2 sequences were identified using a 32P-labeled oligonucleotide probe from near the 5Ј end of the AE2a mRNA (complementary to nt ϩ7 to ϩ22 as numbered in top panel of Fig. 7). Five EcoRI fragments of clone 10-1 that hybridized with the AE2a cDNA, with sizes of 10.2, 4.9, 4.3, 2.1, and 1.8 kb, were subcloned into a plasmid vector and sequenced by the chain termination method using primers corresponding to sequences of the AE2a cDNA [10] or the AE2b and AE2c RACE products. The probe common to all AE2 mRNAs was a SacI-PvuII fragment, corresponding to nt ϩ1118 to ϩ2001 of the AE2a coding sequence [10], that was labeled with [␣-32P]dCTP using random hexamers and the Klenow fragment of DNA polymerase I
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