BRCA1-Dependent Translational Regulation in Breast Cancer Cells

Abstract

BRCA1 (Breast Cancer 1) has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair and protein ubiquitination. We previously demonstrated that BRCA1 interacts with PABP1 (Poly(A)-Binding Protein 1) and that BRCA1 modulates protein synthesis through this interaction. To identify the mRNAs that are translationally regulated by BRCA1, we used a microarray analysis of polysome-bound mRNAs in BRCA1-depleted and non-depleted MCF7 cells. Our findings show that BRCA1 modifies the translational efficiency of approximately 7% of the mRNAs expressed in these cells. Further analysis revealed that several processes contributing to cell surveillance such as cell cycle arrest, cell death, cellular growth and proliferation, DNA repair and gene expression, are largely enriched for the mRNAs whose translation is impacted by BRCA1. The BRCA1-dependent translation of these species of mRNAs therefore uncovers a novel mechanism through which BRCA1 exerts its onco-suppressive role. In addition, the BRCA1-dependent translation of mRNAs participating in unexpected functions such as cellular movement, nucleic acid metabolism or protein trafficking is indicative of novel functions for BRCA1. Finally, this study contributes to the identification of several markers associated with BRCA1 deficiency and to the discovery of new potential anti-neoplastic therapeutic targets.

Figure 1. BRCA1 is a ribosome-associated protein.

A/MCF7 cells were lysed in 25 mM KCl buffer and the post-mitochondrial cytoplasmic lysate was layered onto a 1 M sucrose cushion and centrifuged as described in the “Material and methods” section. Immunoblotting for BRCA1 using the MS110 antibody was performed on the following samples: initial total cell lysate (L), nuclear fraction (N), cytoplasmic fraction (C) and ribosome pellet (R). PABP1 and eIF4G were used as markers for pellet fraction containing ribosome-associated proteins. The analyzed L, N and C fractions represent 5% of the total cell lysate. B/MCF7 cells were lysed in 25 mM KCl buffer and the cytoplasmic fraction was separated onto a 10–40% sucrose gradient. (Top) A characteristic ribosome profile. (Middle) Extracts of total RNA from half of each fraction were subjected to gel analysis to determine the presence of 18S and 28S rRNAs. rRNAs were detected by Gel Red staining. (Bottom) The remaining half of each fraction was precipitated with TCA. BRCA1 protein was identified with immunoblot analysis using D9 antibody. PABP1 and eIF4G served as controls.

Figure 2. Microarray analysis of polysome-associated RNAs from MCF7 cells in which BRCA1 has been depleted.

A/Western blot confirming siRNA inhibition of BRCA1 levels in MCF7 cells when compared with control siRNA. Immunoblotting for BRCA1 used 8F7 antibody. α-tubulin served as loading control. B/Number of mRNAs exhibiting altered translational efficiency in BRCA1-depleted MCF7 cells compared to control MCF7 cells. The 1151 mRNAs displaying a modified relative translatability (RR = polyRNA/totRNA) were clustered in several groups depending on their fold change in polysomal RNA abundance (PolyRNA) and their fold change in total mRNA abundance (TotRNA). The fold changes in polysomal RNA abundance and in total mRNA abundance are indicated as follows: ≤0.67, (↗)≥1.50, (↔) >0.67 and <1.50. The RRs are annotated with a sign and a number. The sign specifies the RR value: (−) ≤0.67, (+) ≥1.50. The number indicates how many mRNAs are deregulated. : mRNAs translationally deregulated through change in polysome mRNA abundance only; : mRNAs translationally deregulated through change in total mRNA abundance only; : mRNAs translationally deregulated through change in polysome abundance together with opposite changes in total mRNA C/Functional distribution of differentially translated known genes in BRCA1-depleted versus control MCF7 cells. Gene functions were established based on the annotation provided by the IPA database. The number of genes enriched in each function is shown in brackets.

Figure 3. RT-qPCR analyses of differentially translated mRNAs upon BRCA1 depletion.

Total RNA and polysomal-associated RNA from MCF7 cells transfected with BRCA1-targetting siRNA or control siRNA were reverse transcribed and five transcripts identified in the microarray analysis were quantified by real time PCR. qPCR analysis was performed in triplicate. Analysis of mRNA levels for each target was normalized to HPRT1 mRNA. For each gene, the polyRNA (grey) and the totalRNA (black) Ratios were determined using the ΔΔCt calculation method. Results are representative of the average RNA ratio ± SEM from four independent experiments. *, p<0.05 compared with SiControl.

Figure 4. Analyses of HIPK2 protein predicted to be modified by BRCA1 depletion.

A/MCF7 cells transfected with BRCA1-targetting siRNA or control siRNA and total protein extracts were collected 72 h later and subjected to immunoblot analysis for HIPK2 protein, using antibodies provided by Epitomics (Left) and Aviva (Right). B/293T cells transfected with BRCA1-targetting siRNA or control siRNA (Left) or transfected with BRCA1 expressing plasmid or empty plasmid as control (Right) were subjected to immunoblot analysis for HIPK2 protein, using antibodies provided by Epitomics. Blots used β-actin and/or α-tubulin as loading controls. The blots shown are representative of at least three independent experiments.