Promoter-sharing by different genes in human genome--CPNE1 and RBM12 gene pair as an example

Abstract

Background: Regulation of gene expression plays important role in cellular functions. Co-regulation of different genes may indicate functional connection or even physical interaction between gene products. Thus analysis on genomic structures that may affect gene expression regulation could shed light on the functions of genes.

Results: In a whole genome analysis of alternative splicing events, we found that two distinct genes, copine I (CPNE1) and RNA binding motif protein 12 (RBM12), share the most 5' exons and therefore the promoter region in human. Further analysis identified many gene pairs in human genome that share the same promoters and 5' exons but have totally different coding sequences. Analysis of genomic and expressed sequences, either cDNAs or expressed sequence tags (ESTs) for CPNE1 and RBM12, confirmed the conservation of this phenomenon during evolutionary courses. The co-expression of the two genes initiated from the same promoter is confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) in different tissues in both human and mouse. High degrees of sequence conservation among multiple species in the 5'UTR region common to CPNE1 and RBM12 were also identified.

Conclusion: Promoter and 5'UTR sharing between CPNE1 and RBM12 is observed in human, mouse and zebrafish. Conservation of this genomic structure in evolutionary courses indicates potential functional interaction between the two genes. More than 20 other gene pairs in human genome were found to have the similar genomic structure in a genome-wide analysis, and it may represent a unique pattern of genomic arrangement that may affect expression regulation of the corresponding genes.

Figure 1

Gene structure of RBM12 and CPNE1 and sharing of promoter and non-coding exons between the two genes in human, mouse and zebrafish. A. Human. Representative cDNAs are [GenBank: NM_152927] for CPNE1, and [GenBank: NM_006047] for RBM12; B. Mouse. Representative cDNAs are [GenBank: NM_170588] for CPNE1, and [GenBank: NM_029397] for RBM12; C. zebrafish. There are two copies for each gene in the zebrafish genome, arranged in a head-to-head direction. The duplicated copies of the genes in zebrafish may pose problem to knock-out experiments in the species. D. partial sequence alignment between zebrafish RBM12 [GenBank: EB783076] and zebrafish CPNE1 [GenBank: NM_199699], labelled as "copine III, like" in NCBI) for the non-coding exon 1 and succeeding sequences. The one nucleotide difference (indicated by arrow) could be due to either polymorphism on the site or a sequencing error. Other sequences that share first exon with [GenBank: EB783076], and therefore may also share promoters with CPNE1 cDNA [GenBank: NM_199699] include: [GenBank: EB832449, DT271069, DT222776 and EB775439] etc.

Figure 2

Phylogenetics analysis of expansion of the CPNE and RBM12 families. Protein sequences of the two gene families from various species were aligned using ClustalX. The aligned sequences were analyzed by MrBayes 3.1.2 for their phylogenetic distances and displayed by TreeView. The numbers shown on each branch are the posterior probabilities of the phylogenetic relationship. The circle marked the genes that shared promoters between CPNE1 and RBM12.

Figure 3

Reverse-Transcription PCR for the co-expression of RBM12 and CPNE1 in mouse tissues and human PBMC. A. Expression of the two genes in various mouse tissues. Top panel (mCPNE1): CPNE1 expression detected by CPNE1-specific primer pair; Second panel (mRBM12): PCR results from RBM12-specific primers; third panel(Com-mCPNE1): amplification from common forward primer from the non-coding exon2 and CPNE1-specific reverse primer from exon 4, the two bands reflect alternative splicing forms including/excluding exon 3; Lower panel(Com-mRBM12): PCR from common forward primer from non-coding exon2 and RBM12-specific primer from the RBM12-specific region of exon 3. B. Expression of the two genes in Human PBMC. Lane 1, 3, 5, 7, 9 are expression of CPNE1 from five individual blood donors, as amplified by a common forward primer in exon1 and CPNE1-specific reverse primer in exon 4. Lane 2, 4, 6, 8, and 10 are expression of RBM12 from the five individuals as amplified by common forward primer from exon 1 and a RBM12-specific primer in exon 3. The different bands in CPNE1 amplification reflect alternative splicing forms.

Figure 4

Alternative splicing forms of CPNE1 and RBM12 in human, mouse and zebrafish in the 5'UTR. GenBank accession nos. for representative cDNA or EST sequences for Human CPNE1: 1. [GenBank:NM_152930], 2. [GenBank:NM_152931], 3. [GenBank:NM_152927], 4. [GenBank:NM_152928], 5. [GenBank:NM_152925]; Human RBM12: 1. [GenBank:NM_006047], 2. [GenBank:NM_152838], 3. [GenBank:AB018308]; mouse CPNE1: 1. [GenBank:CF742269], 2. [GenBank:CN693202], 3. [GenBank:NM_170588], 4. [GenBank:NM_170590]; mouse RBM12: 1. [GenBank:BC052473], 2. [GenBank:AF393216]; zebrafish CPNE1: 1. [GenBank:XM_001338967], 2. [GenBank:EB992764], 3. [GenBank:XM_696989]; zebrafish RBM12: 1. [GenBank:DT275702], 2. [GenBank:EB783076].

Figure 5

Sequence alignment of conserved regions upstream of the coding sequence of CPNE1 and RBM12 among multiple species. A. conservation of exon 1; B. conservation of exon 2; C. conservation in both the splicing acceptor region of intron2 and the non-coding sequence of exon 3.