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Nucleic Acids Res. 2014 Mar;42(5):3177-93. doi: 10.1093/nar/gkt1311. Epub 2013 Dec 17.

NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins.

Author information

1
Institute of Biochemistry I, Medical Faculty, Center for Molecular Medicine Cologne (CMMC), 50931 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, Institute of Genetics, University of Cologne, 50931 Cologne, Germany and Leibniz-Institute of Freshwater Ecology and Inland Fisheries, IGB, Müggelseedamm 301, 12587 Berlin, Germany.

Abstract

NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.

PMID:
24353314
PMCID:
PMC3950704
DOI:
10.1093/nar/gkt1311
[Indexed for MEDLINE]
Free PMC Article

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