Irma Sánchez

Cep76, a Centrosomal Protein that Specifically Restrains Centriole Reduplication

Centrosomes duplicate only once per cell cycle, but the controls that govern this process are largely unknown. We have identified Cep76, a centriolar protein that interacts with CP110. Cep76 is expressed at low levels in G1 and is induced in S and G2 phase, during which point centrioles have already commenced duplication. Interestingly, depletion of Cep76 drives the accumulation of centriolar intermediates in certain types of cancer cells. Enforced Cep76 expression specifically inhibits centriole amplification in cells undergoing multiple rounds of duplication without preventing the formation of extra procentrioles from a parental template. Furthermore, elevated levels of Cep76 do not affect normal centriole duplication. Thus, Cep76 helps limit duplication to once per cell cycle. Our findings also point to mechanistic differences between normal duplication and aberrant centriole amplification, as well as distinctions between diverse modes of amplification.

Cilium assembly and disassembly

The primary cilium is an antenna-like, immotile organelle present on most types of mammalian cells, which interprets extracellular signals that regulate growth and development. Although once considered a vestigial organelle, the primary cilium is now the focus of considerable interest. We now know that ciliary defects lead to a panoply of human diseases, termed ciliopathies, and the loss of this organelle may be an early signature event during oncogenic transformation. Ciliopathies include numerous seemingly unrelated developmental syndromes, with involvement of the retina, kidney, liver, pancreas, skeletal system and brain. Recent studies have begun to clarify the key mechanisms that link cilium assembly and disassembly to the cell cycle, and suggest new possibilities for therapeutic intervention.

SCAPER, a novel cyclin A-interacting protein that regulates cell cycle progression.

Cyclin A/Cdk2 plays an important role during S and G2/M phases of the eukaryotic cell cycle, but the mechanisms by which it regulates cell cycle events are not fully understood. We have biochemically purified and identified SCAPER, a novel protein that specifically interacts with cyclin A/Cdk2 in vivo. Its expression is cell cycle independent, and it associates with cyclin A/Cdk2 at multiple phases of the cell cycle. SCAPER localizes primarily to the endoplasmic reticulum. Ectopic expression of SCAPER sequesters cyclin A from the nucleus and results specifically in an accumulation of cells in M phase of the cell cycle. RNAi-mediated depletion of SCAPER decreases the cytoplasmic pool of cyclin A and delays the G1/S phase transition upon cell cycle re-entry from quiescence. We propose that SCAPER represents a novel cyclin A/Cdk2 regulatory protein that transiently maintains this kinase in the cytoplasm. SCAPER could play a role in distinguishing S phase– from M phase–specific functions of cyclin A/Cdk2.

Tethering of an E3 ligase by PCM1 regulates the abundance of centrosomal KIAA0586/Talpid3 and promotes ciliogenesis

To elucidate the role of centriolar satellites in ciliogenesis, we deleted the gene encoding the PCM1 protein, an integral component of satellites. PCM1 null human cells show marked defects in ciliogenesis, precipitated by the loss of specific proteins from satellites and their relocation to centrioles. We find that an amino-terminal domain of PCM1 can restore ciliogenesis and satellite localization of certain proteins, but not others, pinpointing unique roles for PCM1 and a group of satellite proteins in cilium assembly. Remarkably, we find that PCM1 is essential for tethering the E3 ligase, Mindbomb1 (Mib1), to satellites. In the absence of PCM1, Mib1 destabilizes Talpid3 through poly-ubiquitylation and suppresses cilium assembly. Loss of PCM1 blocks ciliogenesis by abrogating recruitment of ciliary vesicles associated with the Talpid3-binding protein, Rab8, which can be reversed by inactivating Mib1. Thus, PCM1 promotes ciliogenesis by tethering a key E3 ligase to satellites and restricting it from centrioles. DOI: http://dx.doi.org/10.7554/eLife.12950.001

Traf7, a MyoD1 transcriptional target, regulates nuclear factor‐κB activity during myogenesis

We have identified the E3 ligase Traf7 as a direct MyoD1 target and show that cell cycle exit—an early event in muscle differentiation—is linked to decreased Traf7 expression. Depletion of Traf7 accelerates myogenesis, in part through downregulation of nuclear factor‐κB (NF‐κB) activity. We used a proteomic screen to identify NEMO, the NF‐κB essential modulator, as a Traf7‐interacting protein. Finally, we show that ubiquitylation of NF‐κB essential modulator is regulated exclusively by Traf7 activity in myoblasts. Our results suggest a new mechanism by which MyoD1 function is coupled to NF‐κB activity through Traf7, regulating the balance between cell cycle progression and differentiation during myogenesis.

RNF11 sequestration of the E3 ligase SMURF2 on membranes antagonizes SMAD7 down-regulation of transforming growth factor β signaling

The activity of the E3 ligase, SMURF2, is antagonized by an intramolecular, autoinhibitory interaction between its C2 and Hect domains. Relief of SMURF2 autoinhibition is induced by TGFβ and is mediated by the inhibitory SMAD, SMAD7. In a proteomic screen for endomembrane interactants of the RING-domain E3 ligase, RNF11, we identified SMURF2, among a cohort of Hect E3 ligases previously implicated in TGFβ signaling. Reconstitution of the SMURF2·RNF11 complex in vitro unexpectedly revealed robust SMURF2 E3 ligase activity, with biochemical properties previously restricted to the SMURF2·SMAD7 complex. Using in vitro binding assays, we find that RNF11 can directly compete with SMAD7 for SMURF2 and that binding is mutually exclusive and dependent on a proline-rich domain. Moreover, we found that co-expression of RNF11 and SMURF2 dramatically reduced SMURF2 ubiquitylation in the cell. This effect is strictly dependent on complex formation and sorting determinants that regulate the association of RNF11 with membranes. RNF11 is overexpressed in certain tumors, and, importantly, we found that depletion of this protein down-regulated gene expression of several TGFβ-responsive genes, dampened cell proliferation, and dramatically reduced cell migration in response to TGFβ. Our data suggest for the first time that the choice of binding partners for SMURF2 can sustain or repress TGFβ signaling, and RNF11 may promote TGFβ-induced cell migration.

Cell biology: Short RNAs and shortness of breath

The simultaneous deletion of six RNA molecules in mice has been found to cause respiratory and fertility defects, owing to improper assembly of structures called cilia on the cell surface. See Article p.115 The six microRNAs (miRNAs) of the miR-34/449 family are highly similar. Their functions overlap, so it is difficult to determine their specific biological roles. Lin He and colleagues present data obtained from mice deficient for all six miR-34/449 miRNAs. These animals exhibited frequent postnatal mortality, respiratory dysfunction — resembling respiratory complications seen in some patients with primary cilia dyskinesia — and infertility. The main underlying defects were shorter and fewer cilia in the cells of the respiratory tract, largely due to defects in basal body docking to the apical membrane. Of the 57 potential targets of miR-34/449 miRNAs that the authors identify, one is Cp110, a centriolar protein that suppresses cilia assembly. Interestingly, aberrant Cp110 levels in association with respiratory illness have already been uncovered.

Prognostic role of elevated mir-24-3p in breast cancer and its association with the metastatic process

MicroRNAs have been shown to play important roles in breast cancer progression and can serve as biomarkers. To assess the prognostic role of a panel of miRNAs in breast cancer, we collected plasma prospectively at the time of initial diagnosis from 1,780 patients with stage I-III breast cancer prior to definitive treatment. We identified plasma from 115 patients who subsequently developed distant metastases and 115 patients without metastatic disease. Both groups were matched by: age at blood collection, year of blood collection, breast cancer subtype, and stage. The median follow up was 3.4 years (range, 1-9 years). We extracted RNA from plasma and analyzed the expression of 800 miRNAs using Nanostring technology. We then assessed the expression of miRNAs in primary and metastatic breast cancer samples from The Cancer Genome Atlas (TCGA). We found that, miR-24-3p was upregulated in patients with metastases, both in plasma and in breast cancer tissues. Patients whose primary tumors expressed high levels of miR-24-3p had a significantly lower survival rate compared to patients with low mir-24-3p levels in the TCGA cohort (n=1,024). RNA-Seq data of the samples with the highest miR-24-3p expression versus those with the lowest miR-24-3p in the TCGA cohort identified a specific gene expression signature for those tumors with high miR-24-3p. Possible target genes for miR-24-3p were predicted based on gene expression and binding site, and their effects on cancer pathways were evaluated. Cancer, breast cancer and proteoglycans were the top three pathways affected by miR-24-3p overexpression.

Short RNAs and shortness of breath

The simultaneous deletion of six RNA molecules in mice has been found to cause respiratory and fertility defects, owing to improper assembly of structures called cilia on the cell surface. See Article p.115 The six microRNAs (miRNAs) of the miR-34/449 family are highly similar. Their functions overlap, so it is difficult to determine their specific biological roles. Lin He and colleagues present data obtained from mice deficient for all six miR-34/449 miRNAs. These animals exhibited frequent postnatal mortality, respiratory dysfunction — resembling respiratory complications seen in some patients with primary cilia dyskinesia — and infertility. The main underlying defects were shorter and fewer cilia in the cells of the respiratory tract, largely due to defects in basal body docking to the apical membrane. Of the 57 potential targets of miR-34/449 miRNAs that the authors identify, one is Cp110, a centriolar protein that suppresses cilia assembly. Interestingly, aberrant Cp110 levels in association with respiratory illness have already been uncovered.

Short RNAs and shortness of breath: Cell biology

The simultaneous deletion of six RNA molecules in mice has been found to cause respiratory and fertility defects, owing to improper assembly of structures called cilia on the cell surface. See Article p.115 The six microRNAs (miRNAs) of the miR-34/449 family are highly similar. Their functions overlap, so it is difficult to determine their specific biological roles. Lin He and colleagues present data obtained from mice deficient for all six miR-34/449 miRNAs. These animals exhibited frequent postnatal mortality, respiratory dysfunction — resembling respiratory complications seen in some patients with primary cilia dyskinesia — and infertility. The main underlying defects were shorter and fewer cilia in the cells of the respiratory tract, largely due to defects in basal body docking to the apical membrane. Of the 57 potential targets of miR-34/449 miRNAs that the authors identify, one is Cp110, a centriolar protein that suppresses cilia assembly. Interestingly, aberrant Cp110 levels in association with respiratory illness have already been uncovered.