Figure 4.
Identification of proteins associated with treRNA. (A) Top protein candidates from mass spectrometry analysis are listed. The fold ratios of spectral counts of protein candidates in cells expressing MS2-tagged treRNA or MS2 tag control vector were calculated. ∞ indicates that these proteins were detected only in cells expressing treRNA, but not in cells expressing a vector control. (B–E) The components of treRNA RNP complex are required for treRNA functions. (B) The effects on E-cadherin expression in MCF7 cells expressing treRNA by the knockdown of the protein candidates from mass spectrometry analysis. Knockdown of hnRNP K, FXR1, FXR2, and PUF60 expression by two different short-hairpin RNAs (shRNAs) in MCF7-treRNA cells rescued the suppression of E-cadherin by treRNA. (C) The effects on E-cadherin expression in MCF7 cells by the knockdown of the protein candidates. Knockdown of hnRNP K, FXR1, FXR 2, BUB3, TRIM25 and hnRNP E1 expression individually in MCF7 cells does not affect E-cadherin expression. Knockdown of PUF60 expression partially affects E-cadherin expression. Knockdown of SF3B3 expression significantly decreases E-cadherin expression, suggesting that SF3B3 is required for E-cadherin expression in MCF7 cells. (D) MCF7-luc-treRNA cells expressing shRNAs targeting hnRNP K, FXR1, and FXR2 individually or in combination were transplanted into mouse mammary fat pads. The number of mice that developed lung metastasis was reduced when hnRNP K or FXR2 expression was knocked down. No lung metastasis was developed when both hnRNP K and FXR2 expressions were knocked down, or hnRNP K, FXR1, and FXR2 triple expression knockdown. (E) Lung metastatic tumour signals were measured using a luminescence Xenogen imaging system. In the mice that developed lung metastases, tumour signals in the lung were significantly reduced when hnRNP K or FXR2 expression was knocked down when compared with that of cells expressing a control shRNA. There is no lung metastasis signal when both hnRNP K and FXR2 expressions were knocked down, or in the hnRNP K, FXR1 and FXR2 triple expression knockdown. The graph showed the mean photon flux ±s.d. of three independent experiments.
Source data for this figure is available on the online supplementary information page.
