RPL41, a small ribosomal peptide deregulated in tumors, is essential for mitosis and centrosome integrity

Abstract

Ribosomal large subunit protein RPL41 is a basic (positively charged) peptide consisting of only 25 amino acids. An antisense-based functional screening revealed that the down-regulation of RPL41 led to an anchorage-independent growth of NIH3T3 cells in soft agar plates. RPL41 depletion with gene-specific small interfering RNA also resulted in malignant transformation of NIH3T3 cells including increased tumor growth in mice. RPL41 deletion was detected in 59% of tumor cell lines by fluorescence in situ hybridization analyses and RPL41 down-regulation in 75% of primary breast cancers by real-time quantitative reverse transcription-polymerase chain reaction. These studies suggest a tumor suppression role for RPL41. By mass spectrometry, RPL41 was associated with several cytoskeleton components including tubulin beta, gamma, and myosin IIA, which was confirmed by Western blot analysis on both cellular lysis and individually in vitro-expressed proteins. RPL41 also bound directly to polymerized tubulins. Cells overexpressing a GFP-RPL41 were resistant to nocodazole-induced microtubule depolymerization. A synthetic RPL41 induced cellular alpha-tubulin acetylation and G(2)/M cell cycle arrest. These results indicate a stabilizing role of RPL41 on microtubule. Microtubule spindles are essential for chromosome segregation during mitosis. Cells with RPL41 knock-down showed abnormal spindles, frequent failure of cytokinesis, and formation of polynuclear cells. In interphase cells, RPL41-depleted cells had premature splitting of centrosome. Our results provide evidence that RPL41 is a microtubule-associated protein essential for functional spindles and for the integrity of centrosome and that the abnormal mitosis and disrupted centrosome associated with the RPL41 down-regulation may be related to malignant transformation.

Figure 1

Down-regulation of RPL41 in NIH3T3 cells resulted in malignant transformation. (A) RPL41-specific siRNA effectively knocked down cellular RPL41 expression. NIH3T3 cells were transfected with two RPL41 siRNA expression constructs or a control construct expressing a random hairpin siRNA. Western blot analysis was performed with antibodies specific to RPL41 or β-actin. (B) RPL41-depleted NIH3T3 cells formed foci in liquid culture. No such foci were noted in control cells. (C) RPL41-depleted NIH3T3 cells formed significantly more colonies than did control cells in soft agar plates. (D) Colonies that exceeded 120 µm in diameter in soft agar plates were counted. The number of colonies was the average value based on six wells (35 mm). Both cell lines expressing RPL41 siRNA had significantly more colonies than control cells.

Figure 2

Deletion and down-regulation of RPL41 in tumors. (A) RPL41 allelic reduction was detected in human tumors. FISH analysis was performed with an RPL41 BAC clone (red) and a chromosome 12-centromere control probe (green). FISH on normal lymphocytes showed that RPL41 was located at 12q13 as expected (left). Leukemia cells (CCL246) had two copies of centromere and one copy of RPL41 (right). FISH results on 22 tumor cell lines were detailed in Table W1. (B) RPL41 expression was significantly decreased in primary breast cancers. Quantitative real-time RT-PCR was performed with RPL41-specific primers in 12 cases of matched primary breast cancers/normal pairs. Data represent mean ± SD from three reactions. Clinical information of tumors is detailed in Table W2. Constitutional RPL41 inactivation in case 9 could not be excluded.

Figure 3

Interaction of RPL41 with cytoskeletal components. (A) Cellular tubulin α, β, and γ and myosin IIA were effectively pulled down by GST-RPL41. Cell lysate was incubated with GST, GST/RPL41, GST/Scrambled RPL41, and GST/Arginine-rich peptide. Western blot analyses of the GST pull-down products were performed with antibodies to tubulin α, β, and γ and myosin IIA. (B) Cell lysate was incubated with an antibody to RPL41 or rabbit preimmunization serum (control), pulled down by incubation with protein A/G agarose, and immunoblotted with antibodies to tubulin α, β, and γ, myosin IIA, and RPL41. (C) Individually expressed tubulin β and γ and N-terminal myosin IIA were effectively pulled down by GST-RPL41. Proteins were expressed in reticulocyte lysate and labeled with ³⁵S methionine. Myosin IIA was expressed in two overlapping fragments (N-myosin and C-myosin) owing to its large size. (D) RPL41 associated with polymerized tubulin. A synthetic RPL41 peptide was incubated with purified tubulin in the presence or absence of Taxol. Reactions were centrifuged, and the supernatant and precipitants were separated in 20% polyacrylamide gel.

Figure 4

Acetylation of cellular α-tubulin and G₂/M cell cycle arrest induced by RPL41. (A) Significantly increased α-tubulin acetylation was detected in cells treated with a synthetic RPL41. 293T cells and HeLa cells were incubated with 50 µM synthetic RPL41 or vehicle control (saline) for 1 hour and Western blotted with antibodies to α-tubulin, acetyl-α-tubulin, and β-actin. (B and C) Cells overexpressing a GFP-RPL41 showed significantly more residual acetylated α-tubulin after nocodazole challenge than those overexpressing a GFP control. HeLa cells were transfected with GFP (B) or GFP-RPL41 (C) for 24 hours, treated with 10 µM nocodazole or vehicle control (DMSO) for 20 minutes, immunofluorescence-stained with anti-acetyl-α-tubulin antibody (red), and counterstained with DAPI (blue). (D) Cells treated with a synthetic RPL41 accumulated at G₂/S phase. 293T cells were incubated with 25 to 50 µM RPL41 or vehicle (saline) for 16 hours and subjected to a flow cytometry assay. Significant increase in G₂/M cells was seen in cells treated with RPL41.

Figure 5

Cellular localization of RPL41 on human fibroblasts. Immunofluorescence staining of human fibroblasts was performed with antibodies to RPL41, nucleolar marker B23, or α-tubulin, and counterstained with DAPI. (A) In interphase cells, RPL41 is located in nucleoli, nuclei, and cytoplasm. Nucleolar localization of RPL41 was verified by costaining with anti-B23. (B) RPL41 was colocalized with microtubule in the pseudopodia of cells. (C) In mitotic cells, RPL41 was diffused in the entire cell in prophase, concentrated on chromosomes in metaphase, enriched in chromosomes and midzone in anaphase, and localized in newly formed nuclei and midbody in telophase. (D) RPL41 was colocalized with α-tubulin in midbody.

Figure 6

Abnormal microtubules in NIH3T3 cells with RPL41 down-regulation. (A) RPL41-depleted interphase cells showed sparse cellular microtubules and lack of typical aster structure. NIH3T3 stable cell lines expressing an RPL41-specific siRNA (RPL41 KD) or a control random siRNA were immunostained with an antibody to α-tubulin and counterstained with DAPI. (B) RPL41-depleted cells showed abnormal mitotic spindles. Mitotic cells of control and RPL41 KD cells were immunostained with an antibody to α-tubulin and counterstained with DAPI. Control cells had spindles focused on centrosomes, seen as prominent bright foci (arrowheads). These foci were undetectable in RPL41-depleted cells. RPL41-depleted cells also showed lack of midzone spindles in anaphase. (C) Lagged DNA materials were commonly seen in the midbodies in RPL41-depleted cells. (D) Increased polynuclear cells were seen in RPL41-depleted NIH3T3 cells. Approximately 80% decrease in cellular RPL41 in cells expressing a RPL41-specific siRNA was verified by Western blot analysis.

Figure 7

Centrosome abnormality in NIH3T3 cells with RPL41 down-regulation. (A) Majority of RPL41-depleted cells had premature centrosome splitting. Interphase cells of NIH3T3 cells expressing an RPL41-specific siRNA (RPL41 KD) or a control random siRNA were immunostained with an antibody to γ-tubulin and counterstained with DAPI. Control cells had one signal with some consisting of a doublet (insert). Most RPL41-depleted cells had two signals (arrowhead) without doublet (insert). (B) Only one of the split centrioles was-positive to Cep170 in RPL41-depleted cells. Control and RPL41 KD cells were immunostained with antibodies to γ-tubulin and Cep170 (red) and counterstained with DAPI. (C) Defective microtubule regrowth in cells with RPL41 down-regulation. RPL41 KD cells and control cells were subjected to a microtubule regrowth assay followed by immunostaining with antibodies to α-tubulin and γ-tubulin. Typical microtubule asters were seen in control cells but not in RPL41-depleted cells with split centrosomes.