Mechanistic Control of Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM1) Splice Isoforms by the Heterogeneous Nuclear Ribonuclear Proteins hnRNP L, hnRNP A1, and hnRNP M

Kenneth J Dery,Shikha Gaur,Marieta Gencheva,Yun Yen,John E Shively,Rajesh K Gaur

doi:10.1074/jbc.m110.204057

Abstract

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.

Highlights

Maintenance of cell polarity and tissue architecture is vital to the normal growth and differentiation program of epithelial cells [1]
Identification and Characterization of Exon Regulatory Element (ERE) Controlling Alternative Splicing of Exon 7—We recently showed that breast cancer specimens displayed expression of both short and long cytoplasmic domain splice variants of Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) as compared with normal breast tissues that predominately express the S-isoform, suggesting that alteration of CEACAM1 splicing could be a common feature of breast cancer
We demonstrated that regulatory cis-acting elements in exon 7 play a role in the alternative splicing of CEACAM1-L and CEACAM1-S [16]

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Transfection—ZR75 cells were cultured in RPMI 1640 medium, and MDA-MB-468 cells were cultured in minimum Eagle’s medium (MEM) supplemented with 1% sodium pyruvate, 0.15% sodium bicarbonate, 1 ϫ non-essential amino acids, and 1 ϫ penicillin-streptomycin-amphotericin B. 2.5 ␮l of 4 ␮g/␮l heparin and 2.5 ␮l of 5 ϫ loading dye containing 1 ϫ Tris borate EDTA (89 mM Tris-HCl, 89 mM boric acid, 2.5 mM EDTA), 20% glycerol, 0.25% bromphenol blue, and 0.25% xylene cyanol were added, and 3-␮l aliquots of each reaction mixture were loaded onto a 2% horizontal low-melting agarose gel followed by the separation of RNA-containing complexes at 70 V for 4 – 6 h in Tris-glycine running buffer at room temperature [38]. Proteins were boiled for 5 min in cracking buffer (80 mM Tris-Cl, pH 6.8, 0.1 M dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromphenol blue), and separated on 12.5% SDS-polyacrylamide gels. Endogenous hnRNP A1 and hnRNP U (Santa Cruz Biotechnology, 3G6) were visualized with a 1:1000 dilution using an Olympus IX81 Inverted microscope with Q Imaging Retiga 2000R cooled CCD camera with Image Pro Plus Version 6.3 imaging software and 40ϫ magnification

RESULTS

DISCUSSION

Methods