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. 2009 Jul 13;186(1):27-40.
doi: 10.1083/jcb.200810180.

The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability

Shobbir Hussain  1 , Sandra Blanco Benavente, Elisabete Nascimento, Ilaria Dragoni, Agata Kurowski, Astrid Gillich, Peter Humphreys, Michaela Frye
Affiliations

The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability

Shobbir Hussain et al. J Cell Biol. .

Abstract

Myc-induced SUN domain-containing protein (Misu or NSun2) is a nucleolar RNA methyltransferase important for c-Myc-induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA-protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP.

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Figures

Figure 1.
Figure 1.
Misu is nucleolar in interphase and associates with tubulin at the spindle in mitosis. (A) Misu (green) colocalizes with NPM1 (red) at the nucleolus (arrows) but not at the spindle (arrowheads). (B) In prophase, Misu was found at pericentriolar material (arrowheads) but not the spindle fibers (α-tubulin; arrows). (C and D) In metaphase and anaphase, Misu colocalizes with α-tubulin along the spindle (arrows). (E) In telophase, Misu and α-tubulin localize to the midbody (arrows). DNA is counterstained with DAPI (blue). Bars, 10 µm.
Figure 2.
Figure 2.
Misu coimmunoprecipitates with α-tubulin only in mitosis. (A and B) Misu reciprocally coimmunoprecipitates with α-tubulin in HCC1954 cells. (C) Misu only associates with α-tubulin in M phase of the cell cycle. No interaction can be detected when Misu is depleted by RNAi (Misu RNAi). Actin was used a loading control. WCL, whole cell lysate; WB, Western blot. The arrow indicates the size of the proteins.
Figure 3.
Figure 3.
Depletion of Misu in HCC1954 cells results in impaired spindle formation. (A) Cells infected with an Scr RNAi display normal mitosis. (B–D) Misu RNAi results in several adverse effects on mitosis. Chromosomes extend beyond the spindle poles (B, arrows), multipolar spindles (C, arrowheads), individual chromosomes that are misaligned (arrows), and those that are pulled away from the main body of chromosomes (D, arrows) in anaphase. (E) Number of micronuclei in interphase cells increased. Cells are stained for α-tubulin (red) and DAPI (blue). Bar, 10 µm. (F and G) Quantification of effects of Misu RNAi in mitotic cells (F) and interphase cells (G) compared with Scr RNAi. (H) Automated quantification of number of micronuclei in Misu RNAi and Scr RNAi cells. Dotted lines indicate the typical size range of micronuclei. Error bars indicate mean ± SD.
Figure 4.
Figure 4.
Depletion of Misu directly induces mitotic defects. (A–C) Synchronized HCC1954 and Cal51 cells transfected with control and Misu siRNA in the first cell cycle after depletion of Misu. Cells were analyzed after a 45-min recovery period after nocodazole arrest. Depletion of Misu by siRNA (Misu siRNA) causes formation of multipolar spindles (arrowheads) and misaligned chromosomes (arrows). Cells are stained for α-tubulin (A, red) and DAPI (B, blue). (D) Quantification of effects of Misu siRNA in mitotic HCC1954 cells. (E) Control and Misu siRNA cells accumulate to a similar extent in M phase after nocodazole block. (F and G) In Cal51 cells, Misu (green) localizes to nucleoli in interphase and the spindle in mitosis. DNA is counterstained with DAPI. (H) Quantification of effects of Misu siRNA in mitotic Cal51 cells. Chr, Chromosomes. Error bars indicate mean ± SD. Bars, 10 µm.
Figure 5.
Figure 5.
Apoptosis but not necrosis is increased in response to Misu depletion. (A) Flow cytometry to measure cell death and apoptosis using FLICA polycaspase probes in HCC1954 cells infected with Scr RNAi (top), Misu RNAi (middle), and cells treated with actinomycin D as control (bottom). (B and C) Apoptosis is increased in cells with Misu RNAi (B), but necrosis is unchanged (C), as measured by staining with propidium iodide before FACS analysis.
Figure 6.
Figure 6.
Identification of 18S rRNA as binding partner of Misu at nucleoli and the spindle. (A) IP using a Misu antibody or preimmune serum followed by an RNA isolation. RNA with the size of 1.9 kb binds to Misu (left, asterisks) and is degraded in response to RNaseA (right, asterisks). (B) Gene-specific PCR of reverse-transcribed purified RNA identifies 18S rRNA (top) but not 5S or 5.8S rRNA (bottom) as binding partner of Misu. (C and D) IP and RT-PCR in untreated and puromycin-treated cells (C) and unsynchronized versus cells synchronized in G1 and M phase of the cell cycle (D). (E, left) Localization of 18S rRNA (green) using an Alexa Fluor 488–conjugated DNA probe at the nucleoli (asterisk) and the spindle (arrows). (middle and right) Treatment with RNaseA depletes 18S rRNA from the spindle (+RNase). (F and G) Treatment with RNaseA depletes Misu from the spindle but not the spindle poles (arrow). Cells are stained with Misu (green), α-tubulin (red), and DAPI (blue). (H) Quantification of RNase effect on localization of Misu to the spindle. Error bars indicate mean ± SD. Bars, 10 µm.
Figure 7.
Figure 7.
Treatment with RNaseA disrupts spindle morphology, and localization of 18S rRNA to the spindle is independent of active translation. (A–C) Spindle shows diffuse arrangement when cells were treated with RNaseA compared with control. Chromosomes extend beyond the spindle poles (arrowhead), and individual chromosomes are misaligned (arrows). (D–F) Localization of 18S rRNA (green) at the spindles (arrows) is not affected by treatment with puromycin compared with cells treated with PBS (control). Cells are counterstained with α-tubulin (red) and DAPI (blue). Error bars indicate mean ± SD. Bars, 10 µm.
Figure 8.
Figure 8.
The function of Misu at the spindle is independent from its methyltransferase activity. (A) Western blot showing similar protein levels of wild-type Misu (mouse and human) as well as mutant constructs C271A, C321A, D268A, and K190M after purification. Lysates from uninfected cells served as negative control. The asterisk marks a shift in molecular mass in C271A. (B) Human Misu but none of the mutant constructs methylate tRNA in vitro. (C) Western blot showing Misu expression in Scr or Misu RNAi cells coinfected with empty vector control (pB-Empty) or rescue constructs pB-Misu-R, pB-K190M-R, and pB-D268A-R. (D) Quantification of mitotic defects (abnormal spindle) in Misu RNAi cells coinfected with pB-Empty or rescue constructs. Note that all RNAi-resistant constructs rescue defects in spindle morphology (normal mitosis) in the presence of Misu RNAi. Abnormal spindle structure, misaligned chromosomes, or multipolar spindles (Fig. 3) are grouped as abnormal mitosis. (E) Immunofluorescence for tubulin (red) and DAPI (blue) of cells quantified in D. Error bars indicate mean ± SD. Bar, 10 µm.
Figure 9.
Figure 9.
Interaction of Misu and NuSAP is required for localization of NuSAP to the spindle and identification of NuSAP as direct target gene of c-Myc. (A–C) In metaphase of control cells (Scr RNAi), NuSAP (green) colocalizes with α-tubulin (red) to the spindle (arrowheads) and spindle poles (arrows). (right) NuSAP staining at the spindle but not spindle poles is lost in Misu RNAi cells. (D–F) Colocalization of NuSAP with NPM1 at the nucleoli (arrows) is not affected in Misu RNAi cells. DNA is counterstained with DAPI (blue). (G and H) Quantification of A–F. (I) Western blot showing protein levels of NuSAP and Misu in Scr RNAi and Misu RNAi cells synchronized in M phase of the cell cycle. Actin served as loading control. (J and K) Reciprocal co-IP of Misu and NuSAP in HCC1954 cells. (J) NuSAP but not importin-α or -β co-IP with Misu. Preimmunoserum served as negative control (control). WB, Western blot. Arrows mark the size of the indicated proteins. (L) ChIP for NuSAP and nucleolin (NCL) promoters using Myc antibody in epidermis of transgenic (TG) and wild-type (WT) mice. Nucleolin promoters served as positive control, and genomic DNA downstream of the nucleolin promoter served as negative control. Values are normalized to whole cell lysates. (M and N) RNA levels of Misu (M) and NuSAP (N) are elevated in epidermis of Myc-overexpressing transgenic (MycER) but not wild-type mice in response to 4-OHT. Days of treatment are indicated on the x axis. Error bars indicate mean ± SD. Bars, 10 µm.
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