REIIBP methylates nucleolar proteins and regulates pre-rRNA processing

Abstract

The NSD2 (nuclear receptor binding SET [Suppressor of variegation 3-9, Enhancer of zeste, and Trithorax] domain protein 2) gene encodes a short isoform, REIIBP (interleukin-5 response element II binding protein), via alternative transcription initiation. It is particularly overexpressed in t(4;14)+ multiple myeloma. However, the biological functions of REIIBP remain elusive. Here, we show that REIIBP localizes at the dense fibrillar component of the nucleolus. The disordered regions of REIIBP recognize pre-rRNA, which mediates the nucleolar localization of REIIBP. Proteomic analyses reveal that REIIBP is associated with many pre-rRNA processing factors and mediates lysine methylation on a set of pre-rRNA processing factors. Moreover, REIIBP dysregulates pre-rRNA processing, mediated by both disordered regions and the SET domain, and ultimately affects ribosome biogenesis. Collectively, these findings uncover the role of REIIBP in the regulation of ribosome biogenesis, which may drive the pathological process in multiple myeloma.

Keywords: REIIBP; nucleolus; pre-rRNA; protein methylation.

Figure 1

REIIBP localizes in the DFC of the nucleolus. A, schematic diagram of MMSET and REIIBP. B, REIIBP does not colocalize with MMSET. SFB-tagged MMSET or REIIBP were stably expressed in HeLa cells and examined by immunofluorescence (IF) with anti-FLAG antibody. The nucleus and nucleoli were indicated by DAPI staining. Image bar represents 5 μm. C, REIIBP exists in DFC. REIIBP (red) and indicated nucleolar markers (green) were examined by structured illumination microscopy (SIM) in HeLa cells. RPA194, FBL, and NPM1 were used as markers for FC, DFC, and GC, respectively. The fluorescence intensity on the white dashed lines was plotted (lower panels). Scale bar represents 1 μm. DAPI, 4′,6-diamidino-2-phenylindole; DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; HMG box, high mobility group box domain; MMSET, multiple myeloma SET domain; PHD, plant homeodomain zinc finger; PWWP, named for a conserved Pro–Trp–Trp–Pro motif; REIIBP, interleukin-5 response element II binding protein; SET, Suppressor of variegation 3–9, Enhancer of zeste, and Trithorax.

Figure 2

The disordered regions of REIIBP determine the nucleolar localization of REIIBP. A, schematic for REIIBP domain–deletion mutants (left panel). The expression of full-length REIIBP and its domain-deletion mutants in HeLa cells was examined by WB assay (right panel). B, deletion of either disordered regions abolishes the nucleolar localization of REIIBP. The GFP-tagged full-length REIIBP and its domain-deletion mutants were expressed in HeLa cells and examined by IF. Image bar represents 5 μm. The percentage of the cells with positive GFP signal in nucleolus was calculated (n = 150) (lower panel). ∗∗∗p < 0.001, ns, not significant. IF, immunofluorescence; REIIBP, interleukin-5 response element II binding protein; WB, Western blotting.

Figure 3

REIIBP interacts with pre-rRNA. A, the procedure of PAR-CLIP and RNA-Seq using HeLa cells stably expressing SFB-REIIBP (left panel). The RNA species isolated from the cells are shown (right panel). B, the rRNA enriched from HeLa cells stably expressing SFB-REIIBP is compared with that from HeLa cells stably expressing SFB-vector alone. The relative fold of rRNA enrichment is calculated as the percentage of rRNA reads among the reads on all peaks detected by RNA-Seq times the absolute amount of total RNA purified in the PAR-CLIP assays. C, validation of the interaction between REIIBP and pre-rRNA. Following PAR-CLIP assay, RT–qPCR was performed to examine REIIBP-bound pre-rRNAs. ∗∗∗p < 0.001. D, diagrammatic drawing of in vitro GST pull-down-qPCR assay (left panel). Recombinant REIIBP protein binds pre-rRNA in vitro (right panel). GST or GST-REIIBP proteins were incubated with total RNA extracted from HeLa cells. RT–qPCR was performed to examine REIIBP-associated pre-rRNAs using primers targeted on 5′ETS, ITS1, and ITS2 regions of pre-rRNA. ∗∗∗p < 0.001. E, schematics of the REIIBP protein domain structure and its truncated mutants are shown (left panel). The disordered regions of REIIBP bind to pre-rRNA (right panel). Recombinant truncated REIIBP variants were incubated with total RNA extracted from HeLa cells. In vitro GST pull-down-qPCR was performed to determine the interaction between truncated REIIBP variants and pre-rRNA. ∗∗∗p < 0.001, ns, not significant. 5′ETS, 5′ external transcribed spacer; GST, glutathione-S-transferase; ITS, internal transcribed spacer; PAR-CLIP, photoactivatable ribonucleoside–enhanced crosslinking and immunoprecipitation; qPCR, quantitative PCR; REIIBP, interleukin-5 response element II binding protein.

Figure 4

REIIBP associates with rRNA processing–related proteins. A, schematic diagram of immunoprecipitation (IP)–MS experimental process. B, a volcano plot of REIIBP-associated proteins identified by IP–MS. Proteins with significant upregulation (log₂FC ≥1 and p < 0.05) are highlighted in light blue, and rRNA processing–related proteins are marked in light red. Proteins without significant changes (log₂FC <1 and p ≥ 0.05) are shown in gray. The dashed lines indicate the thresholds for statistical significance and FC. C–E, the REIIBP-associated proteins are classified by biological process (C), molecular function (D), and cellular component (E). F, the KEGG pathway analysis of the REIIBP-associated proteins. FC, fold change; IP, immunoprecipitation; KEGG, Kyoto Encyclopedia of Genes and Genomes; MS, mass spectrometry; REIIBP, interleukin-5 response element II binding protein.

Figure 5

REIIBP methylates pre-rRNA–processing proteins. A, LC–MS/MS analysis of NO proteins from HeLa cells stably expressing REIIBP reveals increased lysine methylation in rRNA processing proteins. A total of 75 methylated lysine residues (including mono-, di-, and trimethylated forms) across 40 rRNA processing proteins were identified as having elevated levels. These proteins were systematically classified into seven functional categories. The figure visualizes significant methylation changes with fold change >1.3. B, the chord diagram displays the specific pre-rRNA processing process involving 38 methylation-upregulated candidate proteins. REIIBP, interleukin-5 response element II binding protein.

Figure 6

REIIBP regulates pre-rRNA processing. A, schematic diagram of simplified pre-rRNA processing pathway and the major pre-rRNA intermediates in human cells. The purple, dark red, and dark green lines indicate the location of the 5′ETS, ITS1, and ITS2 hybrid probes, respectively. The lavender, light red, and light green strips cover the pre-rRNA intermediates that the corresponding probe can detect. B, REIIBP dysregulates pre-rRNA processing. NB analyses were performed to examine pre-rRNA intermediates in HeLa cells stably expressing SFB-Vector or SFB-REIIBP (left panel). Indicated probes are shown. The relative levels of 47S, 45S, 30S, 21S, 18S-E, 32S, 12S, and 7S pre-rRNAs are measured (right panel). Values are means ± SD of 3 independent assays. ∗∗∗p < 0.001, ns, not significant. C, disordered regions and the SET domain of REIIBP are required for the dysregulated pre-rRNA processing. NB analyses were performed with probes targeting the 5′ETS, ITS1, and ITS2. D, polysome profiles and polysome-to-monosome (P/M) ratio change upon REIIBP expression. Data are presented as mean ± SD (n = 3); ∗p < 0.05. 5′ETS, 5′ external transcribed spacer; ITS, internal transcribed spacer; L/E, long exposure; NB, Northern blotting; REIIBP, interleukin-5 response element II binding protein; S/E, short exposure; SET, Suppressor of variegation 3–9, Enhancer of zeste, and Trithorax.