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Tissue-specific alternative splicing of TCF7L2.

Prokunina-Olsson L, Welch C, Hansson O, Adhikari N, Scott LJ, Usher N, Tong M, Sprau A, Swift A, Bonnycastle LL, Erdos MR, He Z, Saxena R, Harmon B, Kotova O, Hoffman EP, Altshuler D, Groop L, Boehnke M, Collins FS, Hall JL - Hum. Mol. Genet. (2009)

Bottom Line: Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146).After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant.GenBank Accession Numbers: FJ010164-FJ010174.

View Article: PubMed Central - PubMed

Affiliation: Genome Technology Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA. prokuninal@mail.nih.gov

ABSTRACT
Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r(2) = 0.84-0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164-FJ010174.

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Structure of the N-terminal part of TCF7L2: location of the β-catenin binding domain encoded by exons 1 and 2, and a T2D-associated LD block with SNPs rs7903146 and rs12255372. Shown: constitutive exons—black rectangles, alternative exons—white rectangles; alternative transcription starts sites TSS1, TSS2 and TSS3—arrows, potential translation starts—‘ATG’.
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DDP321F3: Structure of the N-terminal part of TCF7L2: location of the β-catenin binding domain encoded by exons 1 and 2, and a T2D-associated LD block with SNPs rs7903146 and rs12255372. Shown: constitutive exons—black rectangles, alternative exons—white rectangles; alternative transcription starts sites TSS1, TSS2 and TSS3—arrows, potential translation starts—‘ATG’.

Mentions: Since our analysis did not reveal any association between expression of TCF7L2 measured by assay ‘ex7–8’ and SNPs rs7903146 and rs12255372, we hypothesized that these genetic variants might be associated with alternative splicing. Members of the TCF/LEF family of transcription factors use alternative transcription start sites (TSSs) to produce functionally distinct proteins (42–44). To identify all TSS of TCF7L2, we performed 5′ rapid amplification of cDNA ends (5′RACE) in an RNA pool of eight tissues (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscles, subcutaneous adipose tissue and lymphoblastoid cell lines). We identified several TSS: (1) TSS1, located at −536 bp from the first translation start site and corresponding to RefSeq sequence (NM_030756); (2) TSS2, located at position +83 bp within exon 1; (3) a cluster collectively referred as TSS3 located in the first intron of the gene (+200, +239, +302, +338 bp, Fig. 3 and Supplementary Material, Table S2). Only transcripts initiated from the TSS1 can be translated from a start codon within exon 1 and produce protein isoforms with the ß-catenin binding domain encoded by exons 1 and 2. The next in-frame translation start site that can be used by transcripts expressed from the TSS2 and TSS3, is located within exon 3 (Fig. 3), and the resulting proteins would lack the ß-catenin binding domain. Both the main (exon 1) and the alternative translation start sites (exon 3) have weak Kozak consensus sequences, ‘aaaaaaATGC’ and ‘ttcatcATGA’, compared with a classical consensus site ‘gcca/gccATGG’ (45). We also performed 3′RACE in an attempt to detect alternative splicing forms of the 3′-UTRs. We did not detect any short PCR fragments corresponding to mRNA transcripts with significantly truncated 3′-UTR (data not shown). We PCR amplified, cloned and sequenced full-length cDNA fragments starting from the first translation start site within exon 1 through the end of the transcript in exon 14 (based on RefSeq NM_030756). We did not attempt to clone full-length cDNAs initiated from the TSS2 because this transcription start is embedded within exon 1 of the TSS1 form and the products would represent a mixture of transcripts. Because of low expression from the TSS3, further study of the transcripts initiated from this TSS was not pursued in detail. Sequencing of full-length cDNA clones reconfirmed several known alternative exons (3a, 4a, 12, 13, 13a and 13b) and minor in-frame inclusions of 3, 12 and 15 bp in exons 4a, 6 and 8, respectively (39–41) (Supplementary Material, Table S2). All sequences for full-length cDNA expression constructs were deposited in the GenBank (FJ010164–FJ010174, Supplementary Material, Table S3).


Tissue-specific alternative splicing of TCF7L2.

Prokunina-Olsson L, Welch C, Hansson O, Adhikari N, Scott LJ, Usher N, Tong M, Sprau A, Swift A, Bonnycastle LL, Erdos MR, He Z, Saxena R, Harmon B, Kotova O, Hoffman EP, Altshuler D, Groop L, Boehnke M, Collins FS, Hall JL - Hum. Mol. Genet. (2009)

Structure of the N-terminal part of TCF7L2: location of the β-catenin binding domain encoded by exons 1 and 2, and a T2D-associated LD block with SNPs rs7903146 and rs12255372. Shown: constitutive exons—black rectangles, alternative exons—white rectangles; alternative transcription starts sites TSS1, TSS2 and TSS3—arrows, potential translation starts—‘ATG’.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748888&req=5

DDP321F3: Structure of the N-terminal part of TCF7L2: location of the β-catenin binding domain encoded by exons 1 and 2, and a T2D-associated LD block with SNPs rs7903146 and rs12255372. Shown: constitutive exons—black rectangles, alternative exons—white rectangles; alternative transcription starts sites TSS1, TSS2 and TSS3—arrows, potential translation starts—‘ATG’.
Mentions: Since our analysis did not reveal any association between expression of TCF7L2 measured by assay ‘ex7–8’ and SNPs rs7903146 and rs12255372, we hypothesized that these genetic variants might be associated with alternative splicing. Members of the TCF/LEF family of transcription factors use alternative transcription start sites (TSSs) to produce functionally distinct proteins (42–44). To identify all TSS of TCF7L2, we performed 5′ rapid amplification of cDNA ends (5′RACE) in an RNA pool of eight tissues (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscles, subcutaneous adipose tissue and lymphoblastoid cell lines). We identified several TSS: (1) TSS1, located at −536 bp from the first translation start site and corresponding to RefSeq sequence (NM_030756); (2) TSS2, located at position +83 bp within exon 1; (3) a cluster collectively referred as TSS3 located in the first intron of the gene (+200, +239, +302, +338 bp, Fig. 3 and Supplementary Material, Table S2). Only transcripts initiated from the TSS1 can be translated from a start codon within exon 1 and produce protein isoforms with the ß-catenin binding domain encoded by exons 1 and 2. The next in-frame translation start site that can be used by transcripts expressed from the TSS2 and TSS3, is located within exon 3 (Fig. 3), and the resulting proteins would lack the ß-catenin binding domain. Both the main (exon 1) and the alternative translation start sites (exon 3) have weak Kozak consensus sequences, ‘aaaaaaATGC’ and ‘ttcatcATGA’, compared with a classical consensus site ‘gcca/gccATGG’ (45). We also performed 3′RACE in an attempt to detect alternative splicing forms of the 3′-UTRs. We did not detect any short PCR fragments corresponding to mRNA transcripts with significantly truncated 3′-UTR (data not shown). We PCR amplified, cloned and sequenced full-length cDNA fragments starting from the first translation start site within exon 1 through the end of the transcript in exon 14 (based on RefSeq NM_030756). We did not attempt to clone full-length cDNAs initiated from the TSS2 because this transcription start is embedded within exon 1 of the TSS1 form and the products would represent a mixture of transcripts. Because of low expression from the TSS3, further study of the transcripts initiated from this TSS was not pursued in detail. Sequencing of full-length cDNA clones reconfirmed several known alternative exons (3a, 4a, 12, 13, 13a and 13b) and minor in-frame inclusions of 3, 12 and 15 bp in exons 4a, 6 and 8, respectively (39–41) (Supplementary Material, Table S2). All sequences for full-length cDNA expression constructs were deposited in the GenBank (FJ010164–FJ010174, Supplementary Material, Table S3).

Bottom Line: Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146).After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant.GenBank Accession Numbers: FJ010164-FJ010174.

View Article: PubMed Central - PubMed

Affiliation: Genome Technology Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA. prokuninal@mail.nih.gov

ABSTRACT
Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r(2) = 0.84-0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164-FJ010174.

Show MeSH
Related in: MedlinePlus