APC/C-mediated degradation of dsRNA-binding protein 4 (DRB4) involved in RNA silencing

Background

Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.

Methodology/Principal Findings

In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.

Conclusions/Significance

Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4.     

PKD controls mitotic Golgi complex fragmentation through a Raf–MEK1 pathway

Before entering mitosis, the stacks of the Golgi cisternae are separated from each other, and inhibiting this process delays entry of mammalian cells into mitosis. Protein kinase D (PKD) is known to be involved in Golgi-to–cell surface transport by controlling the biogenesis of specific transport carriers. Here we show that depletion of PKD1 and PKD2 proteins from HeLa cells by small interfering RNA leads to the accumulation of cells in the G2 phase of the cell cycle and prevents cells from entering mitosis. We further provide evidence that inhibition of PKD blocks mitotic Raf-1 and mitogen-activated protein kinase kinase (MEK) activation, and, as a consequence, mitotic Golgi fragmentation, which could be rescued by expression of active MEK1. Finally, Golgi fluorescence recovery after photobleaching analyses demonstrate that PKD is crucial for the cleavage of the noncompact zones of Golgi membranes in G2 phase. Our findings suggest that PKD controls interstack Golgi connections in a Raf-1/MEK1–dependent manner, a process required for entry of the cells into mitosis.     

A mutation-promotive role of nucleotide excision repair in cell cycle-arrested cell populations following UV irradiation

Growing attention is paid to the concept that mutations arising in stationary, non-proliferating cell populations considerably contribute to evolution, aging, and pathogenesis. If such mutations are beneficial to the affected cell, in the sense of allowing a restart of proliferation, they are called adaptive mutations. In order to identify cellular processes responsible for adaptive mutagenesis in eukaryotes, we study frameshift mutations occurring during auxotrophy-caused cell cycle arrest in the model organism Saccharomyces cerevisiae. Previous work has shown that an exposure of cells to UV irradiation during prolonged cell cycle arrest resulted in an increased incidence of mutations. In the present work, we determined the influence of defects in the nucleotide excision repair (NER) pathway on the incidence of UV-induced adaptive mutations in stationary cells. The mutation frequency was decreased in Rad16-deficient cells and further decreased in Rad16/Rad26 double-deficient cells. A knockout of the RAD14 gene, the ortholog of the human XPA gene, even resulted in a nearly complete abolishment of UV-induced mutagenesis in cell cycle-arrested cells. Thus, the NER pathway, responsible for a normally accurate repair of UV-induced DNA damage, paradoxically is required for the generation and/or fixation of UV-induced frameshift mutations specifically in non-replicating cells.     

Radiocadmium exchange with seawater by Fundulus heteroclitus (L.) (Pisces: Cyprinodontidae)

Mummichogs, Fundulus heteroclitus (L.) accumulated Cd 115m (NO₃)₂ from synthetic seawater solutions during a period of 25 days, but the rate of whole body accumulation decreased with increasing concentrations of stable Cd²⁺ in the medium. Viscera were the major repository of Cd 115m during uptake, especially gastrointestinal tract and to a lesser extent liver. Loss in whole body radioactivity over a post-treatment interval of 180 days in cadmium-free seawater was about 90%, with gallbladder and especially liver most influential in the excretion processes; this pattern was unchanged by the level of stable Cd²⁺ present in the medium during uptake. The significance of this and other observations in terms of field monitoring of cadmium utilizing teleosts as indicators is discussed.     

Non-homologous end joining dependency of gamma-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by gamma-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the gamma-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.     

Diagnosis of cattle diseases endemic to sub-Saharan Africa: evaluating a low cost decision support tool in use by veterinary personnel

Background

Diagnosis is key to control and prevention of livestock diseases. In areas of sub-Saharan Africa where private practitioners rarely replace Government veterinary services reduced in effectiveness by structural adjustment programmes, those who remain lack resources for diagnosis and might benefit from decision support.

Methodology/Principal Findings

We evaluated whether a low-cost diagnostic decision support tool would lead to changes in clinical diagnostic practice by fifteen veterinary and animal health officers undertaking primary animal healthcare in Uganda. The eight diseases covered by the tool included 98% of all bovine diagnoses made before or after its introduction. It may therefore inform proportional morbidity in the area; breed, age and geographic location effects were consistent with current epidemiological understanding. Trypanosomosis, theileriosis, anaplasmosis, and parasitic gastroenteritis were the most common conditions among 713 bovine clinical cases diagnosed prior to introduction of the tool. Thereafter, in 747 bovine clinical cases estimated proportional morbidity of fasciolosis doubled, while theileriosis and parasitic gastroenteritis were diagnosed less commonly and the average number of clinical signs increased from 3.5 to 4.9 per case, with 28% of cases reporting six or more signs compared to 3% beforehand. Anaemia/pallor, weakness and staring coat contributed most to this increase, approximately doubling in number and were recorded in over half of all cases. Finally, although lack of a gold standard hindered objective assessment of whether the tool improved the reliability of diagnosis, informative concordance and misclassification matrices yielded useful insights into its role in the diagnostic process.

Conclusions/Significance

The diagnostic decision support tool covered the majority of diagnoses made before or after its introduction, leading to a significant increase in the number of clinical signs recorded, suggesting this as a key beneficial consequence of its use. It may also inform approximate proportional morbidity and represent a useful epidemiological tool in poorly resourced areas.     

Electronically Controlled Continuous Culture Device

A photocell-controlled continuous culture device, a Nephelostat, is described that maintains a wide variety of cultures of microorganisms in balanced growth. This Nephelostat controls concentrations of bacteria within +/-3% over a cell concentration range of 10(6) to 10(9) cells per ml. Growth rates are recorded so that changes in the growth rate are observed over small increments of time. Spontaneous and caffeine-induced mutation rates of two strains of Escherichia coli were compared under Nephelostat and chemostat conditions.     

Improved Temperature Control of the Technicon Tissue Processor Paraffin Bath

Modifications of the AutoTechnicon Tissue Processor Paraffin Bath are described. The modifications are a combination of electrical and mechanical changes that will ensure long-term reliability of the unit and dependable temperature maintenance of the paraffin. Extensive testing has verified that modified units are able to stabilize temperature within a range of 0.25C over many months of continuous operation.     

High-Intensity Vapor Lamp for Biological Research

A vapor arc light source has been adapted to the study of the lethal action on bacteria of near-ultraviolet (UV) and visible light. Its use makes possible much shorter exposure times than could be obtained from previously available sources. The output of radiant energy is sufficient to provide a fairly detailed action spectrum for lethality in the long-UV and visible region without the addition of exogenous sensitizers. Populations of cells of Escherichia coli WP2 were inactivated through five log(10) cycles with light at 460 nm. Significant inactivation also was obtained with light at 550 and 650 nm.