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A novel CDX2 isoform regulates alternative splicing.

Witek ME, Snook AE, Lin JE, Blomain ES, Xiang B, Magee MS, Magee M, Waldman SA - PLoS ONE (2014)

Bottom Line: CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2.Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35.These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS).

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kimmel Cancer Center & Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS) that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain). CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS).

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CDX2/AS influences alternative splice site selection of minigenes in 293T and LovoCDX2−/− cells.CDX2/AS regulates alternative splicing of Tra2β-1 and CD44v5 minigenes in Lovo minigenes in LovoCDX2−/− cells independent of re-introduction of CDX2 expression (p0.05). Reverse images of four independent experiments.
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pone-0104293-g006: CDX2/AS influences alternative splice site selection of minigenes in 293T and LovoCDX2−/− cells.CDX2/AS regulates alternative splicing of Tra2β-1 and CD44v5 minigenes in Lovo minigenes in LovoCDX2−/− cells independent of re-introduction of CDX2 expression (p0.05). Reverse images of four independent experiments.

Mentions: We next conducted splicing assays to further define a role of CDX2/AS in pre-mRNA processing. To this end, we obtained a Lovo colorectal cancer cell line devoid of CDX2 expression (LovoCDX2−/−) to test the role of CDX2/AS in alternative splicing in a physiologic setting and examine the impact of CDX2 expression on CDX2/AS splicing activity [23]. We co-transfected LovoCDX2−/− cells with either a Tra2β-1 or CD44v5 minigene with or without CDX2/AS and in the absence and presence of CDX2. CDX2/AS significantly (p0.05) increased exclusion of exon 2 from Tra2β-1 and exon 5 from CD44v5 by 10%, independently of CDX2 expression, confirming the ability of CDX2/AS to alter splice site selection in colon-derived cells (Fig. 6). To evaluate these effects in vivo, T84 xenografts were electroporated with either MSCV-EGFP or MSCV-CDX2/AS-His and the Tra2β-1 minigene. Similar to results observed in LovoCDX2−/− cells in culture, CDX2/AS altered the splicing pattern of the minigene expressed in T84 xenografts by 10% (Fig. S1).


A novel CDX2 isoform regulates alternative splicing.

Witek ME, Snook AE, Lin JE, Blomain ES, Xiang B, Magee MS, Magee M, Waldman SA - PLoS ONE (2014)

CDX2/AS influences alternative splice site selection of minigenes in 293T and LovoCDX2−/− cells.CDX2/AS regulates alternative splicing of Tra2β-1 and CD44v5 minigenes in Lovo minigenes in LovoCDX2−/− cells independent of re-introduction of CDX2 expression (p0.05). Reverse images of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125279&req=5

pone-0104293-g006: CDX2/AS influences alternative splice site selection of minigenes in 293T and LovoCDX2−/− cells.CDX2/AS regulates alternative splicing of Tra2β-1 and CD44v5 minigenes in Lovo minigenes in LovoCDX2−/− cells independent of re-introduction of CDX2 expression (p0.05). Reverse images of four independent experiments.
Mentions: We next conducted splicing assays to further define a role of CDX2/AS in pre-mRNA processing. To this end, we obtained a Lovo colorectal cancer cell line devoid of CDX2 expression (LovoCDX2−/−) to test the role of CDX2/AS in alternative splicing in a physiologic setting and examine the impact of CDX2 expression on CDX2/AS splicing activity [23]. We co-transfected LovoCDX2−/− cells with either a Tra2β-1 or CD44v5 minigene with or without CDX2/AS and in the absence and presence of CDX2. CDX2/AS significantly (p0.05) increased exclusion of exon 2 from Tra2β-1 and exon 5 from CD44v5 by 10%, independently of CDX2 expression, confirming the ability of CDX2/AS to alter splice site selection in colon-derived cells (Fig. 6). To evaluate these effects in vivo, T84 xenografts were electroporated with either MSCV-EGFP or MSCV-CDX2/AS-His and the Tra2β-1 minigene. Similar to results observed in LovoCDX2−/− cells in culture, CDX2/AS altered the splicing pattern of the minigene expressed in T84 xenografts by 10% (Fig. S1).

Bottom Line: CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2.Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35.These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS).

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kimmel Cancer Center & Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS) that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain). CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2) and pre-mRNA processing (CDX2/AS).

Show MeSH
Related in: MedlinePlus