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

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.

Arabidopsis MAS2, an Essential Gene That Encodes a Homolog of Animal NF-κ B Activating Protein, Is Involved in 45S Ribosomal DNA Silencing

“…Our results also agree with those obtained with the mammalian NKAPs, which localize to nuclear speckles and interact with nuclear ribonuclear proteins, splicing factors, several RNA helicases, and other factors involved in the maturation of 45S pre-rRNA and ribosome biogenesis (Burgute et al, 2014).…”

“…Human NKAP interacts with pre-mRNAs and intronless mRNAs; it also interacts with FUSED IN SARCOMA (Burgute et al, 2014), a multifunctional protein involved in the regulation of alternative splicing and mRNA export from the nucleus to the cytoplasm. Taking together the recent results on human NKAP and our study of Arabidopsis MAS2, it seems plausible that NKAPs function in the regulation of the translation of aberrant or unspliced mRNAs, by regulating their nuclear export.…”

“…Human NKAP seems to be a multifunctional protein: It is a component of the Notch corepressor complex, it is required for proper T cell development (Pajerowski et al, 2009), and it plays an unknown role in splicing (Burgute et al, 2014).…”

“…NOTCH proteins are transmembrane receptors involved in several conserved pathways that regulate cellular differentiation, proliferation, and apoptosis in animals (Bray, 2006). Human NKAP has also been found bound to different spliceosomal complexes (Jurica and Moore, 2002;Bessonov et al, 2008Bessonov et al, , 2010Ilagan et al, 2013) and to pre-mRNAs and spliced mRNAs (Burgute et al, 2014). A role has been proposed for human NKAP in regulating constitutive splicing.…”

“…NKAP interacts with several RNA binding proteins, including splicing factors; it binds to exons in pre-mRNAs as well as to small nuclear RNAs, small nucleolar RNAs, rRNAs, and long intergenic noncoding RNAs. XIST (X-inactive-specific transcript) regulatory RNA, the major effector of X chromosome epigenetic inactivation, has also been found bound to NKAP (Burgute et al, 2014).…”