The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle–regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5′ untranslated region (5′ UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

During cell cycle progression in the bacterium Caulobacter crescentus, the master cell cycle regulator CtrA is controlled by CcnA, a cell cycle-regulated non-coding RNA transcribed from a gene located at the origin of replication.     

Glutamate at the phosphorylation site of response regulator CtrA provides essential activities without increasing DNA binding

The essential response regulator CtrA controls the Caulobacter crescentus cell cycle and phosphorylated CtrA~P preferentially binds target DNA in vitro. The CtrA aspartate to glutamate (D51E) mutation mimics phosphorylated CtrA~P in vivo and rescues non-viable C.crescentus cells. However, we observe that the CtrA D51E and the unphosphorylated CtrA wild-type proteins have identical DNA affinities and produce identical DNase I protection footprints inside the C.crescentus replication origin. There fore, D51E promotes essential CtrA activities separate from increased DNA binding. Accordingly, we argue that CtrA protein recruitment to target DNA is not sufficient to regulate cell cycle progression.     

A single hydrophobic cleft in the <Emphasis Type="Italic">Escherichia coli</Emphasis> processivity clamp is sufficient to support cell viability and DNA damage-induced mutagenesis <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The ubiquitous family of DnaN sliding processivity clamp proteins plays essential roles in DNA replication, DNA repair, and cell cycle progression, in part by managing the actions of the different proteins involved in these processes. Interactions of the homodimeric Escherichia coli β clamp with its known partners involves multiple surfaces, including a hydrophobic cleft located near the C-terminus of each clamp protomer.     

CtrA controls cell division and outer membrane composition of the pathogen Brucella abortus

Brucella abortus is a pathogen infecting cattle, able to survive, traffic, and proliferate inside host cells. It belongs to the Alphaproteobacteria, a phylogenetic group comprising bacteria with free living, symbiotic, and pathogenic lifestyles. An essential regulator of cell cycle progression named CtrA was described in the model bacterium Caulobacter crescentus. This regulator is conserved in many alphaproteobacteria, but the evolution of its regulon remains elusive. Here we identified promoters that are CtrA targets using ChIP‐seq and we found that CtrA binds to promoters of genes involved in cell cycle progression, in addition to numerous genes encoding outer membrane components involved in export of membrane proteins and synthesis of lipopolysaccharide. Analysis of a conditional B. abortus ctrA loss of function mutant confirmed that CtrA controls cell division. Impairment of cell division generates elongated and branched morphologies, that are also detectable inside HeLa cells. Surprisingly, abnormal bacteria are able to traffic to the endoplasmic reticulum, the usual replication niche of B. abortus in host cells. We also found that CtrA depletion affected outer membrane composition, in particular the abundance and spatial distribution of Omp25. Control of the B. abortus envelope composition by CtrA indicates the plasticity of the CtrA regulon along evolution.     

Checkpoint effects and telomere amplification during DNA re-replication in fission yeast

Background  

Although much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, Schizosaccharomyces pombe.     

Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast

Background  

Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner.     

Characterization of novel elongated Parvulin isoforms that are ubiquitously expressed in human tissues and originate from alternative transcription initiation

Background  

The peptidyl prolyl cis/trans isomerase (PPIase) Parvulin (Par14/PIN4) is highly conserved in all metazoans and is assumed to play a role in cell cycle progression and chromatin remodeling. It is predominantly localized to the nucleus and binds to chromosomal DNA as well as bent oligonucleotides in vitro.     

An essential protease involved in bacterial cell-cycle control.

Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control. We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle. Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process. ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase. Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential. Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA. CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter. In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.     

Up-regulation of kin17 is essential for proliferation of breast cancer

Background

Kin17 is ubiquitously expressed at low levels in human tissue and participates in DNA replication, DNA repair and cell cycle control. Breast cancer cells are characterized by enabling replicative immortality and accumulated DNA damage. However, whether kin17 contributes to breast carcinogenesis remains unknown.

Methodology/Principal Findings

In this study, we show for the first time that kin17 is an important molecule related to breast cancer. Our results show that kin17 expression was markedly increased in clinical breast tumors and was associated with tumor grade, Ki-67 expression, p53 mutation status and progesterone receptor expression, which were assessed in a clinicopathologic characteristics review. Knockdown of kin17 inhibited DNA replication and repair, blocked cell cycle progression and inhibited anchorage-independent growth, while increasing sensitivity to chemotherapy in breast cancer cells. Moreover, kin17 silencing decreased EGF-stimulated cell growth. Furthermore, overexpression of kin17 promoted DNA replication and cell proliferation in MCF-10A.

Conclusions/Significance

Our findings indicate that up-regulation of kin17 is strongly associated with cellular proliferation, DNA replication, DNA damage response and breast cancer development. The increased level of kin17 was not only a consequence of immortalization but also associated with tumorigenesis. Therefore, kin17 could be a novel therapeutic target for inhibiting cell growth in breast cancer.     

Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1‐S transition involves the degradation of an essential DNA‐binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1‐S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1‐S transition so that cells can properly activate CtrA‐dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.     

The NIMA-family kinase,Nek1 affects the stability of centrosomes and ciliogenesis

Background  

Mutations in Nek1 (NIMA-Related Kinase 1) are causal in the murine models of polycystic kidney disease kat and kat ^2J . The Neks are known as cell cycle kinases, but recent work in protists has revealed that in addition to roles in the regulation of cell cycle progression, some Neks also regulate cilia. In most cells, cilia are disassembled prior to mitosis and are regenerated after cytokinesis. We propose that Neks participate in the coordination of ciliogenesis with cell cycle progression. Mammalian Nek1 is a candidate for this activity because renal cysts form in response to dysfunctional ciliary signalling.