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. 2016 Jan 14;44(5):2348–2361. doi: 10.1093/nar/gkw009

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — The WQHD motif in ALYREF is important for its interactions with spliced mRNA, eIF4A3 and CBP80. (A) The WQHD motif in ALYREF is placed in a low-complexity region. In the ALYREF point mutant used in this study, the WxHD motif is replaced by DQAK. (B) In vitro splicing reactions of MINX substrate were supplemented with extracts expressing FLAG-tagged ALYREF wild-type and DQAK mutant (as in Figure 1A). Splicing products are schematically represented on the left side of a gel. (C) Expression of FLAG-tagged proteins in HEK 293 extracts used in (B) was determined by immunoblot analysis using a FLAG-antibody. (D) FLAG-immunoprecipitations of RNase A-treated lysates from stable cell lines with induced expression of 3x FLAG ALYREF WT and WQHD mutant. Co-immunoprecipitated CBP80, eIF4A3 and UAP56 were detected by immunoblotting using a CBP80-, eIF4A3- and UAP56-specific antibodies, respectively. A total of 5% of cell extracts were loaded as input. Extracts without induced expression of 3xFLAG-tagged proteins were used as controls. (E) Localization of FLAG-emGFP ALYREF mutants in HeLa cells. DAPI was used to stain nuclear DNA. Scale bar, 5 μm.