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. 2015;12(3):255-67.
doi: 10.1080/15476286.2015.1017221.

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

Wen-An Pan  1 , Hsin-Yue Tsai, Shun-Chang Wang, Michael Hsiao, Pei-Yu Wu, Ming-Daw Tsai
Affiliations

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

Wen-An Pan et al. RNA Biol. 2015.

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.

Keywords: 5S RNP, 5S ribonucleoprotein particle; CDK1, cyclin dependent kinase 1; DFC, dense fibrillar component; ETS/ITS, external/internal transcribed spacers; GSK3, glycogen synthase kinase 3; Ki67; Ki67FHAID, Ki67-FHA interaction domain; LSU, large subunit; MDM2, murine double minute 2; NIFK, Nucleolar protein Interacting with the FHA domain of pKi-67; NPM1/B23, nucleophosmin; Noprecipitation; PAR-CLIP, Photo-Activatable-Ribonucleoside-Enhanced Crosslinking and Immu-pre-rRNAs, rRNA precursors; REMSA, RNA electrophoresis mobility shift assay; RNA recognition motif; RNP1 and 2, ribonucleoprotein motif 1 and 2; RPL5 and RPL11, large ribosomal protein 5 and 11; RRM, RNA recognition motif; cell cycle; nucleolar stress; rNIFK, recombinant NIFK; ribosome biogenesis; snoRNP, small nucleolar ribonucleoprotein.

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Figures

Figure 1.
Figure 1.
Silencing of NIFK induces reversible p53 dependent G1 arrest. (A) Cellular proliferation of U2OS cells transfected with siNIFK. (B) Flow cytometry analyses of asynchronous and G2/M synchronous U2OS cells transfected with siNIFK. (C) Same as B, with indicated siRNA alone or in combination. The quantifications of 2 repeats are also shown in the right panel. (D) Western blot analysis of the expressions of NIFK, p53, and p21 in asynchronous U2OS cells transfected with indicated siRNA.
Figure 2.
Figure 2.
NIFK is required for G1 progression by participating in rRNA processing. (A) Flow cytometry analysis of G2/M synchronous U2OS cells transfected with indicated siRNAs (left panel), and Western blot analysis of indicated proteins of asynchronous cells (right panel). (B) Immunofluorescent staining of U2OS cells showing subnucleolar localization of NIFK (green), Ki67 (red), fibrillarin (red), and nuclei (blue). (C) 32P-orthophosphate based pulse-chase analysis showing the kinetics of nascent rRNA synthesis in U2OS cells transfected with siNIFK. 32P labeled RNAs are separated by 1% agarose-formaldehyde gel and visualized by autoradiography (upper panel). The total RNA is shown by ethidium bromide (EtBr) staining (lower panel). (D) The same as (C) except the RNAs were separated by a 10% polyacrylamide-7 M urea gel. The total RNA is shown by ethidium bromide (EtBr) staining (lower panel and full view in Fig. S2D).
Figure 3.
Figure 3.
NIFK-mediated pre-rRNA processing and G1 progression require RRM but not Ki67FHAID. (A) Schematic representation of NIFK functional domains and designing of ectopic NIFK expression constructs (upper panel). For immuno and fluorescent detection, Flag-tag epitope and GFP were fused upstream of NIFK cDNA. dR indicates RRM deletion; dK, Ki67FHAID deletion; TA, T234AT238A; Vec, Flag-GFP vector. The procedure and time line for phenotypic rescue experiments are shown in the bottom panel. (B) Cell proliferation assay for cells phenotypically rescued by NIFK wild-type and mutants. (C) Flow cytometry analyses (left) and quantification (right) of rescued cells after G2/M synchronization. (D) 32P-orthophosphate based pulse-chase analysis showing the nascent rRNA synthesis in phenotypically rescued cells. 32P labeld RNAs at 4.5 h chasing time were visualized by autoradiography (upper panel) and EtBr staining (lower panel). (E) Western blot analysis showing p53 and p21 levels in the phenotypically rescued cells described in (D). Anti-NIFK antibody (NIFK) detects both endogenous and ectopically expressed NIFK. The asterisk indicates ectopic NIFK and the arrow, endogenous NIFK.
Figure 4.
Figure 4.
Identification of specific residues responsible for the RRM function in rRNA processing and G1 progression. (A) Sequence alignment of NIFK-RRM orthologues from different species. Asterisks indicate residues selected for alanine substitution. (B) Cell cycle analyses, (C) nascent rRNA synthesis, and (D) Western blot analyses of the phenotypically rescued cells described in Fig. 3, showing defects of NIFK-RRM mutants in rescuing NIFK silencing. The asterisk indicates ectopic NIFK and the arrow, endogenous NIFK.
Figure 5.
Figure 5.
NIFK regulates 28S and 5.8S rRNA maturation through processing of ITS1 site 2. (A) Schematic representation of human rRNA transcripts. Upper, human 47S precursor rRNA showing the transcribed spacers (ETS and ITS), coding sequences, and cleavage sites. Lower left, illustration of 47S pre-rRNA processing pathways. The alternative steps are colored gray. Lower right, overview of pre-rRNA intermediates. Arrowheads indicate the positions of the probes used in Northern blot analysis. (B-F) Northern blot analyses showing the pre-rRNAs derived from siNIFK transfected U2OS cells. The specific rRNA species were detected using probes complementary to the regions downstream of site A0 of 5′ETS (P1, shown in B), between 18S and site 2a (P2, shown in C), between site 2a and 2 of ITS1 (P3, shown in D), between 5.8S and site 4 of ITS2 (P4, shown in E), and 18S, 5.8S, and 28S rRNA (probes 5, 6 and 7, shown in F). (G) Northern blot analysis of the pre-rRNAs derived from phenotypic rescued cells using probe P4. The complementation of NIFK silencing by NIFK and NIFK-KF mutant following that described in Fig. 4. (H) The same as (E), except that the siRPL5 transfected U2OS cells are also compared.
Figure 6 (See previous page).
Figure 6 (See previous page).
The RRM of NIFK binds to the 5′-end of ITS2 rRNA. (A) Western blot analysis of NIFK associated with indicated RNAs. Biotinylated or non-biotinylated RNAs were immobilized on streptavidin beads. RNA coupling efficiency was shown by SYBR green staining (lower panel). The proteins brought down by RNA were eluted, and analyzed by Western blot using NIFK antibody (upper panel). (B) Northern blot of RNase footprinting of ITS2 1-200 RNA protected by rNIFK. Lane 1, RNase T1 digested RNAs. Several G positions are indicated. Lane 2, RNA ladders generated by alkaline hydrolysis. Lanes 3, 0.016 U/μL RNase I digested RNA. Lanes 4-5, RNase I digested RNA in the absence (R) or presence (R+P) of rNIFK. Lanes 6-7, longer exposure of autoradiography shown in lanes 4-5. The bracket marks the position protected by rNIFK. (C) ITS2 50-150 RNA secondary structure detection. Left panel, autoradiography of 5′-end labeled RNA. Lane 1, RNA ladders generated by alkaline hydrolysis. Lanes 2-3, RNase T1 digested RNA with (+) or without (−) urea. Several G positions are indicated. Lanes 4-6, RNAs digested with increasing amount of RNase A. Right panel, predicted secondary RNA structure. The numbers indicate the positions with respective to ITS2 1-200. (D) REMSA analyses of RNAs bound by rNIFK and rNIFK-4YK in vitro. 32P internal-labeled ITS2 1-200 RNA was incubated with increasing concentration of rNIFK (left panel) or rNIFK-4YK (right panel), separated, and detected by autoradiography. The retarded motility corresponding to RNA-protein complex is indicated. (E) Northern blot analysis of NIFK associated rRNA species. Ectopically expressed proteins were immunoprecipitated by Flag-antibody. 10% of immunoprecipitated proteins were analyzed by Western blot (lower panel), and the rest were subjected to RNA extraction followed by Northern blot analysis using probe P4 (upper panel). The asterisk indicates the antibody heavy chain. A non-specific band appears at 28S.
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