RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J

Abstract

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin‐dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence‐binding protein J (RBP‐J)/CBF‐1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP‐J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP‐J/CBF‐1‐interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP‐J/CBF‐1 from the nucleus. Functionally, we show that RITA can reverse a Notch‐induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch‐mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP‐J/CBF‐1, where its amounts are limiting.

Negative control of CSL gene transcription by stress/DNA damage response and p53

“…For instance, in mammalian cells Wnt signaling can inhibit CSL activity by direct binding of Dishevelled, and CSL stability is regulated by Presenilin-2 and p38 MAP-kinase, while its cellular localization can be regulated in Xenopus laevis or mammalian cells by RITA or SMRT protein complexes. [8][9][10][11] Recent advances in human genomic and transcriptomic analyses provide insights into individual differences in susceptibility to disease, with tissue-specific control of gene expression as key determinant. 12 For novel insights into control of CSL gene expression, we started from the study of individual variations in gene expression in human dermal fibroblasts (HDFs) derived from many different individuals.…”

RBP-J-interacting and tubulin-associated protein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma by activating the p53–Fbxw7 pathway

“…Immunoprecipitation experiments were carried out using whole-cell extracts from SMMC7721 cells 24 h after cotransfection with RITA-Flag [13]. The extracts were incubated with 20 lL protein A/G-agarose beads (Shang Hai Yue-ke Biotechnology CO., LTD) at 4°C for 4 h. After centrifugation, the supernatants were incubated with 100 lL anti-Flag rabbit antibody (1:100) (M2, Sigma) or 100 lL anti-RBP-J mouse antibody (1:100) (Institute of Immunology Co., Ltd), respectively at 4°C for 60 min.…”

“…RITA and control siRNAs were chemically synthesized (GenePharma, Shanghai, China), and the target sequences were designed according to Wacker et al [13]. The siRNAs were delivered into the SMMC7721 and HepG2 cells at 70% confluence using X-tremeGENE siRNA transfection reagent (Roche, Mannheim, Germany) following the manufacturer's protocol.…”

“…The transcription factor RBP-J may play an important role in the regulation of Notch signaling, as it is crucial for recognition of the DNA target sequences in both the repressor and activator complexes. RBP-J-interacting and tubulin-associated (RITA; C12ORF52) is a highly conserved, 36-kDa protein that has no significant homology to any other protein [13]. On a functional level, RITA interferes with Notch-and RBP-J-mediated transcription.…”

From EosFP to mIrisFP: structure‐based development of advanced photoactivatable marker proteins of the GFP‐family

“…A single mitochondrion was photoconverted and its development was monitored as a function of time [42]. For protein tracking, the tubulin-binding protein RITA [52] was fused to mEosFPthermo and expressed at 37 C ( Figure 5C). Consequently, tubulin fibers in a HeLa cell are highlighted by the green fluorescence of mEosFPthermo.…”