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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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Focal nuclear localization of CDX2/AS is RS-domain dependent.(A) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag or MSCV-CDX2/AS-His revealed diffuse and focal nuclear localization patterns for CDX2 and CDX2/AS, respectively, with minimal areas of overlap (dotted circles). (B) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag, MSCV-CDX2/AS-His revealed nuclear localization whereas MSCV-CDX2/ASΔRS-His, a mutant CDX2/AS protein lacking bases distal to the alternatively induced frameshift, resulted in peri-nuclear localization.
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pone-0104293-g002: Focal nuclear localization of CDX2/AS is RS-domain dependent.(A) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag or MSCV-CDX2/AS-His revealed diffuse and focal nuclear localization patterns for CDX2 and CDX2/AS, respectively, with minimal areas of overlap (dotted circles). (B) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag, MSCV-CDX2/AS-His revealed nuclear localization whereas MSCV-CDX2/ASΔRS-His, a mutant CDX2/AS protein lacking bases distal to the alternatively induced frameshift, resulted in peri-nuclear localization.

Mentions: We examined subcellular localization of CDX2 and CDX2/AS in 293T cells transiently transfected with pMSCV-CDX2-Flag and/or pMSCV-CDX2/AS-His by fluorescent immunohistochemistry (F-IHC). Although both proteins localized specifically to the nucleus, CDX2 exhibited a diffuse localization pattern in contrast to the accumulation of CDX2/AS in nuclear foci with minimal areas of co-localization with CDX2 (Fig. 2A). To determine the role of the RS domain of CDX2/AS in its subcellular localization, we performed F-IHC studies on 293T cells transiently transfected with a mutant protein (pMSCV-CDX2/ASΔRS-His) containing only bases proximal to the alternative splicing-induced frameshift. Interestingly, deletion of the RS domain resulted in perinuclear sequestration of the protein suggesting importance of these amino acids in protein stability and trafficking to the nucleus (Fig. 2B).


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)

Focal nuclear localization of CDX2/AS is RS-domain dependent.(A) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag or MSCV-CDX2/AS-His revealed diffuse and focal nuclear localization patterns for CDX2 and CDX2/AS, respectively, with minimal areas of overlap (dotted circles). (B) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag, MSCV-CDX2/AS-His revealed nuclear localization whereas MSCV-CDX2/ASΔRS-His, a mutant CDX2/AS protein lacking bases distal to the alternatively induced frameshift, resulted in peri-nuclear localization.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104293-g002: Focal nuclear localization of CDX2/AS is RS-domain dependent.(A) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag or MSCV-CDX2/AS-His revealed diffuse and focal nuclear localization patterns for CDX2 and CDX2/AS, respectively, with minimal areas of overlap (dotted circles). (B) F-IHC analysis of 293T cells transiently transfected with MSCV-CDX2-Flag, MSCV-CDX2/AS-His revealed nuclear localization whereas MSCV-CDX2/ASΔRS-His, a mutant CDX2/AS protein lacking bases distal to the alternatively induced frameshift, resulted in peri-nuclear localization.
Mentions: We examined subcellular localization of CDX2 and CDX2/AS in 293T cells transiently transfected with pMSCV-CDX2-Flag and/or pMSCV-CDX2/AS-His by fluorescent immunohistochemistry (F-IHC). Although both proteins localized specifically to the nucleus, CDX2 exhibited a diffuse localization pattern in contrast to the accumulation of CDX2/AS in nuclear foci with minimal areas of co-localization with CDX2 (Fig. 2A). To determine the role of the RS domain of CDX2/AS in its subcellular localization, we performed F-IHC studies on 293T cells transiently transfected with a mutant protein (pMSCV-CDX2/ASΔRS-His) containing only bases proximal to the alternative splicing-induced frameshift. Interestingly, deletion of the RS domain resulted in perinuclear sequestration of the protein suggesting importance of these amino acids in protein stability and trafficking to the nucleus (Fig. 2B).

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