The RNA recognition motif of NIFK is required for rRNA maturation during cell cycle progression

Abstract

Ribosome biogenesis governs protein synthesis. NIFK is transactivated by c-Myc, the key regulator of ribosome biogenesis. The biological function of human NIFK is not well established, except that it has been shown to interact with Ki67 and NPM1. Here we report that NIFK is required for cell cycle progression and participates in the ribosome biogenesis via its RNA recognition motif (RRM). We show that silencing of NIFK inhibits cell proliferation through a reversible p53-dependent G1 arrest, possibly by induction of the RPL5/RPL11-mediated nucleolar stress. Mechanistically it is the consequence of impaired maturation of 28S and 5.8S rRNA resulting from inefficient cleavage of internal transcribed spacer (ITS) 1, a critical step in the separation of pre-ribosome to small and large subunits. Complementation of NIFK silencing by mutants shows that RNA-binding ability of RRM is essential for the pre-rRNA processing and G1 progression. More specifically, we validate that the RRM of NIFK preferentially binds to the 5′-region of ITS2 rRNA likely in both sequence specific and secondary structure dependent manners. Our results show how NIFK is involved in cell cycle progression through RRM-dependent pre-rRNA maturation, which could enhance our understanding of the function of NIFK in cell proliferation, and potentially also cancer and ribosomopathies.

Aurora A and NF-κB Survival Pathway Drive Chemoresistance in Acute Myeloid Leukemia via the TRAF-Interacting Protein TIFA

“…All resulting STR profiles were matched with known ATCC fingerprints (www.ATCC.org). Maintenance of respective cell lines followed our previous protocols (21)(22)(23). 293T and HeLa cells were maintained in DMEM(Gibco).…”

“…To perform retroviral transduction, pseudotyped viruses were packaged by the pantropic retroviral expression system in the presence of VSV-G according to the manufacturer's instructions (Clontech) and subjected to infection of indicated cells as described previously (22). After 400 mg/mL G418 selection for weeks, the expressions of ectopic proteins in stable cells were confirmed by Western blot analysis before experimental analysis or siRNA transfection for phenotypic rescue experiments.…”

“…After 48 hours, cells were washed with icecold PBS and subjected to apoptosis detection using FITC Annexin V apoptosis detection kit according to the manufacturer's instructions (BD Biosciences). Cells were then gated for myeloid lineage by light-scattering following a previously described protocol (28), and apoptosis rate, as suggested by double staining of propidium iodide and FITC Annexin V, was analyzed by BD FACSCalibur Flow Cytometer (BD FACSCalibur) as described previously (22). For analysis of U937 cells in the xenograft model, bone marrow cells or mouse PBMCs were sequentially stained with anti-human CD33 FITC and CD45 PerCP/Cy5.5, respectively, for 1 hour on ice.…”

“…To address the molecular mechanism of targeting TIFA, we phenotypically rescued TIFA-depleted AML lines by wild-type TIFA and T9A mutant following a silencing-complementation strategy (22). The silencing-dependent chemosensitivity was efficiently alleviated upon expression of wild-type TIFA, but not the T9A mutant, in all tested AML lines ( Fig.…”

Biogenesis and Function of Small Non-Coding RNAs Derived from Eukaryotic Ribosomal RNA

“…Thus, the heterogeneity found in ribosomes may allow this regulation by favoring translation of specific sets of mRNAs into proteins. Both the type of proteins present in the RNP complex 3,[25][26][27][28] and the rRNAs may contribute to this regulation, as in some alternative pathways during the eukaryotic rRNA maturations 4,21,29 . In addition, splicing of some rRNA transcripts may be decoupled and lead to the production of new rRNA intermediates (e.g.…”