BRCA1-Dependent Translational Regulation in Breast Cancer Cells

<div><p>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.</p></div

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

<p>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.</p

BRCA1 is a ribosome-associated protein.

<p>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 “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067313#s2" target="_blank">Material and methods</a>” 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.</p

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

<p>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. *, <i>p</i><0.05 compared with SiControl.</p

Direct Visualization of the Highly Polymorphic <i>RNU2</i> Locus in Proximity to the <i>BRCA1</i> Gene

<div><p>Although the breast cancer susceptibility gene <i>BRCA1</i> is one of the most extensively characterized genetic loci, much less is known about its upstream variable number tandem repeat element, the <i>RNU2</i> locus. <i>RNU2</i> encodes the U2 small nuclear RNA, an essential splicing element, but this locus is missing from the human genome assembly due to the inherent difficulty in the assembly of repetitive sequences. To fill the gap between <i>RNU2</i> and <i>BRCA1</i>, we have reconstructed the physical map of this region by re-examining genomic clone sequences of public databases, which allowed us to precisely localize the <i>RNU2</i> array 124 kb telomeric to <i>BRCA1</i>. We measured by performing FISH analyses on combed DNA for the first time the exact number of repeats carried by each of the two alleles in 41 individuals and found a range of 6-82 copies and a level of heterozygosity of 98%. The precise localisation of the <i>RNU2</i> locus in the genome reference assembly and the implementation of a new technical tool to study it will make the detailed exploration of this locus possible. This recently neglected macrosatellite could be valuable for evaluating the potential role of structural variations in disease due to its location next to a major cancer susceptibility gene.</p></div

Visualization by molecular combing of the 17q21 region around <i>BRCA1</i>.

<p>(A) Schematization of the genomic morse code used. The <i>BRCA1</i> Genomic Morse Code (GMC) depicted (v4.0) is an improvement of the published code (v1.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Cheeseman1" target="_blank">[23]</a>. It covers a genomic region of 200 kb and consist in 17 signals of a distinct color (green, red or blue), each composed of 1 to 3 small horizontal bars corresponding to a single DNA probe. The signals for the flanking probes FP1-4 are each composed of 2 green or blue horizontal bars, while the signal for the <i>RNU2</i> array repeat unit is composed of 1 red horizontal bar. Of note, the probe for the <i>RNU2</i> array cross-reacts with <i>RNU2-4P</i>. (B) Fourteen fibres displaying different numbers of <i>RNU2</i> signals are shown. The first six fibres display the entire bar code from the <i>BRCA1</i> GMC to <i>RNU2-4P</i>, while the followings miss either the beginning of the <i>BRCA1</i> GMC or <i>RNU2-4P</i>.</p

Localisation of the <i>RNU2</i> macrosatellite within the chromosome 17 sequence assemblies from NCBI Build 37.p10.

<p>(A) Schema of the region surrounding the <i>RNU2</i> array. The location of the portion of the <i>RNU2</i> repeat unit (not comprising the <i>RNU2</i> gene) and of the right junction found in the assemblies are depicted, as well as the probes used in molecular combing experiments that flank the <i>RNU2</i> array (FP1-4), and the <i>NBR1</i> and <i>TMEM106A</i> genes. (B) Clones covering the region. The reference sequence assemblies is based upon the complete sequence of 3 overlapping BACs, RP11-242D8, CTD-3014M21 and RP11-100E5 (AC060780.18, AC109326.11 and AC087650.12 respectively), represented by brown arrows. The complete sequence of the WI2-3095P13 fosmid (AC160862.2, green arrow) matches the reference sequence. The sequence of the ABC10-44487500M2 fosmid (AC231386.2, green arrow) matches the reference sequence up to its centromeric extremity where it contains several <i>RNU2</i> repeat units (depicted in a dotted curl). The five unassembled contigs of the working draft sequence of the RP11-570A16 BAC clone (AC087365.3) showing homology with the reference sequence are represented by a purple arrow. Contig 15 has been mis-assembled, as it contains several <i>RNU2</i> repeat units (depicted in dotted curls) at both its extremities.</p

Visualization by FISH on mitotic metaphase chromosome of the <i>RNU2</i> locus.

<p>Two probes were used, one consisting of the 6.1</p

Description of the <i>RNU2</i> array alleles identified in 46 unrelated chromosomes.

<p>Description of the <i>RNU2</i> array alleles identified in 46 unrelated chromosomes.</p

Schematic representation of the chromosome 17q21 region around the <i>BRCA1</i> gene.

<p>(A) Gene locations and physical map distances as reported in the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Liu1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076054#pone.0076054-Pavelitz2" target="_blank">[19]</a>. (B) Gene locations within a 300 Kb window as shown in the UCSC Genome Browser. Arrows indicate transcription direction. BRCA1P1: <i>BRCA1</i> pseudogene.</p