The RNA binding protein Larp1 regulates cell division, apoptosis and cell migration

Abstract

The RNA binding protein Larp1 was originally shown to be involved in spermatogenesis, embryogenesis and cell-cycle progression in Drosophila. Our data show that mammalian Larp1 is found in a complex with poly A binding protein and eukaryote initiation factor 4E and is associated with 60S and 80S ribosomal subunits. A reduction in Larp1 expression by siRNA inhibits global protein synthesis rates and results in mitotic arrest and delayed cell migration. Consistent with these data we show that Larp1 protein is present at the leading edge of migrating cells and interacts directly with cytoskeletal components. Taken together, these data suggest a role for Larp1 in facilitating the synthesis of proteins required for cellular remodelling and migration.

Figure 1.

(A) Larp proteins are conserved in metazoans and members of the Larp1 family contain an N-terminal La domain (similar to La proteins) and a C-terminal conserved or Larp1 region containing DM15 tandem repeat regions (33). There is a single ‘Larp’ gene in D. melanogaster [now termed DmLarp1 (27)] and two homologues in humans, Larp1 and Larp1b (previously termed Larp2) encoded by genes located at 5q33.2 and 4q28.2, respectively. Larp1 and Larp1b share 50 and 59% identity with Dm Larp1, respectively. There are two isoforms of Larp1 protein of 1019 and 1096 amino acids, respectively, and three isoforms of Larp1b protein of 914, 512 and 358 amino acids. (B) Of the two human Larp proteins, Larp1 is more abundant than Larp1b as shown by qPCR in HeLa cells (after normalization to housekeeping genes). The relative expression level of Larp1b (when standardized to 100% Larp1) was 2.5%, therefore Larp1 is 40-fold more abundant. (C) Immunoprecipitation, using antibodies to Larp1 and PABP and protein G sepharose beads showed an interaction between Larp1 and PABP, as demonstrated using western blotting of the immunoprecipitate. This interaction was not observed using beads alone (control) but was not disrupted following treatment with RNAse A. (D) Western blot of protein pull-down using 7-Methyl-GTP cap-binding sepharose beads demonstrates that Larp1 exists in complex with eIF4E. As expected, PABP and eIF4G were also ‘pulled down’ from the lysate using the cap beads in an association that was not disrupted after RNAse treatment. Additionally, loss of Larp1 (by RNAi) did not remove the interaction between either PABP and eIF4E or eIF4G and eIF4E (data not shown), suggesting these interactions are non-Larp1 dependent.

Figure 2.

(A) Ribosome sedimentation profiles using HeLa cells treated with either Larp1 siRNA (20 and 40 nM), control siRNA (20 nM) or untreated. Compared to controls, the profiles demonstrated a considerable increase in the ratio of the subpolysome components (40S, 60S and 80S) compared to polysomes after Larp1 siRNA (20 and 40 nM) treatment, indicating that loss of Larp1 results in defective translation initiation. (B) Global protein synthesis rates in cells treated with Larp1 siRNA and non-sense siRNA were compared using 35S methionine incorporation and show that there is a 15% reduction in total protein synthesis in cells which lack Larp 1. (C) Western blotting of HeLa cell lysates pre and post Larp1 RNAi probed with antibodies for translation initiation components. Loss of Larp1 did not alter levels of eIF4G, eIF4E or eIF2α, but was associated with increase in levels of hypophosphorylated 4E-BP1. (D) Western blotting following cap pull-down of HeLa lysates pre- and post-Larp1 RNAi showed increased association between eIF4E and 4E-BP1. (E) Western blotting of proteins precipitated from elute fractions obtained during ribosome sedimentation profiling showed a predominant association between Larp1 and the pre-polysome components, the 40S, 60S and 80S ribosome fractions.

Figure 3.

(A) Flow cytometry profile of HeLa cells following Larp1 RNAi treatment (red hatched) compared to control RNAi (black line). In cells treated with Larp1 siRNA, an increase in 4C relative to 2C cells is observed along with an increase in cell death (arrow). RNAi was performed using 20 nM Larp1 siRNA and 20 nM control siRNA. (B) Immunofluorescence showing U2OS cells during mitosis compared to controls (inset). Following knockdown of Larp1 after siRNA treatment, cells have a distinct ‘scattered chromosome’ appearance. Alpha-tubulin (green), γ-tubulin (red) and DNA (blue) (scale bar = 10 µm) compared to controls. (C) In control treated HeLa cells Cyclin B levels are elevated in metaphase (i) but are decreased following anaphase (ii). Following Larp1 RNAi, levels of Cyclin B remain high, showing the characteristic ‘scattered chromosome’ phenotype and without transition to anaphase (iii and iv) (scale bar = 10 µm).

Figure 4.

(A) Immunofluorescence of HeLa cells following treatment with control RNAi, Larp1 RNAi and in cells transfected with Larp1 over-expressing construct (FLAG-Larp1) examined using confocal microscopy. Panels show images in combined colour channel (i), blue channel (DNA) (ii), red channel (actin) (iii) and green channel (Larp1) (iv). Larp1 over-expression (FLAG-Larp1) causes a mesenchymal-like phenotype, with cells displaying multiple lamellipodia (white arrow). Cells treated with Larp1 RNAi were larger and had more filopodia when compared to controls. (B) Scratch migration assay in HeLa cells treated with Larp1 siRNA or control siRNA control. An open furrow was generated by scratching confluent cells using a pipette tip. Confluency was restored in controls between 15 and 18 h. However, in cells treated with Larp1 siRNA, confluency was not restored after 18 h. The distance between furrow edges in the control and Larp1 siRNA treated cells was measured and is presented graphically in (C). In (D), cells were fixed and immunostained 12 h after scratch. Cells at the furrow edge were examined using confocal microscopy. This showed, in cells treated with control RNAi (top panel), a concentration of Larp1 within their leading edge (small arrows in magnified figures) in the direction of cell travel. This was not observed after Larp1 RNAi (bottom panel), where cells were closely adherent to each other without a clear direction of travel.

Figure 4.

(A) Immunofluorescence of HeLa cells following treatment with control RNAi, Larp1 RNAi and in cells transfected with Larp1 over-expressing construct (FLAG-Larp1) examined using confocal microscopy. Panels show images in combined colour channel (i), blue channel (DNA) (ii), red channel (actin) (iii) and green channel (Larp1) (iv). Larp1 over-expression (FLAG-Larp1) causes a mesenchymal-like phenotype, with cells displaying multiple lamellipodia (white arrow). Cells treated with Larp1 RNAi were larger and had more filopodia when compared to controls. (B) Scratch migration assay in HeLa cells treated with Larp1 siRNA or control siRNA control. An open furrow was generated by scratching confluent cells using a pipette tip. Confluency was restored in controls between 15 and 18 h. However, in cells treated with Larp1 siRNA, confluency was not restored after 18 h. The distance between furrow edges in the control and Larp1 siRNA treated cells was measured and is presented graphically in (C). In (D), cells were fixed and immunostained 12 h after scratch. Cells at the furrow edge were examined using confocal microscopy. This showed, in cells treated with control RNAi (top panel), a concentration of Larp1 within their leading edge (small arrows in magnified figures) in the direction of cell travel. This was not observed after Larp1 RNAi (bottom panel), where cells were closely adherent to each other without a clear direction of travel.

Figure 5.

Immunofluorescence of HeLa cells following treatment with control RNAi, Larp1 RNAi and in cells transfected with Larp1 over-expressing construct (FLAG-Larp1) examined using confocal microscopy. Panels show images in combined colour channel (i), green channel (β-actin) (ii), red channel (γ-actin) (iii) and blue channel (DNA) (iv). Stress fibres containing β-actin are present in control cells (white arrows) along with β- and γ-actin colocalization in cortical fibres. After Larp1 RNAi, stress fibres are no longer present and β-actin has a diffusely cytoplasmic localization. Following Larp1 over-expression (FLAG-Larp1) cells again demonstrate a mesenchymal morphology with absent stress fibres but a concentration of γ-actin within lamellipodia (arrowhead).