Dissecting apoptosis the omics way

A combined analysis of transcription, translation and protein degradation reveals the global effects of an anticancer drug on tumour cells

Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor.

Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions

Parents of Children with Complex Trauma: Occupations Viewed Through Photovoice

This qualitative study represents a project idea that shifted focus during the three-year doctoral program. The original idea was to develop an intervention model in response to an identified need within a clinical setting. Specifically, within a clinic that specialized in services for people with sensory processing disorders, a trend was observed that suggested a more traditional sensory processing approach was no longer as effective as it had been in the past. Further investigation into potential factors revealed that the children who did not demonstrate expected improvements presented some common factors. Many of the children were adopted, some from orphanages and some from foster homes. Many, if not most children were identified with trauma histories. Jane Koomar, a well-known expert in sensory integration, and Dan Hughes, a renowned expert in the field of trauma and attachment disorders, developed a conceptual model incorporating sensory integration-based occupational therapy, mental health, and the family. My project in the Evidence Based Practice class addressed the evidence for this approach, in preparation for an anticipated feasibility study. Clinic circumstances necessitated a change, however, and in the Occupation, Participation, and Justice class I interviewed one parent and conducted a focus group of three parents whose children had complex trauma. Parents described feelings of isolation and marginalization, they experienced diminished social circles, and services were difficult to find, even though their families had financial resources to access them. It was clear that the families were affected, not just the children, and in fact parents identified their wish for improved quality of family life. Further literature review suggested that these feelings and experiences were similar to those of parents with children who had mental health difficulties and disabilities in general. Missing from the literature, however, was a broad discussion of the occupations of parents whose children had complex trauma, from their own perspectives. This doctoral project therefore focused on the occupations of parents whose children had complex trauma. While all the course work in the doctoral program helped provide building blocks for this doctoral project, three classes were especially helpful in this regard. The Educational Methods and Practices class furthered my interest in critical theory, extending into theories of learning, especially constructivism. Each person’s perspective is unique, informed by past experiences, and as such, study of parent occupations as individuals was compelling to me. The Evolution of Ideas class subsequently provided the framework for my study of occupation. Wilcock and Towsend’s (2014) conceptualization of occupation as doing, being, becoming, and belonging became the framework for this study. Input from the first Occupation, Participation, and Justice class study, paired with the relatively sparse literature on the topic of occupations of parents whose children have complex trauma, suggested the need and benefit of further study in this area. Specific aims of the current doctoral study are to explore the occupations of this group of parents, and to provide an empowerment opportunity for the parents as they understand and advocate for desired changes in their lives. Results of this current study will hopefully contribute to knowledge and literature in the field of occupational science, and in turn will help inform occupational therapy practice and further research

Global analysis of gene expression reveals mRNA superinduction is required for the inducible immune response to a bacterial pathogen.

The inducible innate immune response to infection requires a concerted process of gene expression that is regulated at multiple levels. Most global analyses of the innate immune response have focused on transcription induced by defined immunostimulatory ligands, such as lipopolysaccharide. However, the response to pathogens involves additional complexity, as pathogens interfere with virtually every step of gene expression. How cells respond to pathogen-mediated disruption of gene expression to nevertheless initiate protective responses remains unclear. We previously discovered that a pathogen-mediated blockade of host protein synthesis provokes the production of specific pro-inflammatory cytokines. It remains unclear how these cytokines are produced despite the global pathogen-induced block of translation. We addressed this question by using parallel RNAseq and ribosome profiling to characterize the response of macrophages to infection with the intracellular bacterial pathogen Legionella pneumophila. Our results reveal that mRNA superinduction is required for the inducible immune response to a bacterial pathogen

The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly.

Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss

Topology and Robustness in the Drosophila Segment Polarity Network

A complex hierarchy of genetic interactions converts a single-celled Drosophila melanogaster egg into a multicellular embryo with 14 segments. Previously, von Dassow et al. reported that a mathematical model of the genetic interactions that defined the polarity of segments (the segment polarity network) was robust (von Dassow et al. 2000). As quantitative information about the system was unavailable, parameters were sampled randomly. A surprisingly large fraction of these parameter sets allowed the model to maintain and elaborate on the segment polarity pattern. This robustness is due to the positive feedback of gene products on their own expression, which induces individual cells in a model segment to adopt different stable expression states (bistability) corresponding to different cell types in the segment polarity pattern. A positive feedback loop will only yield multiple stable states when the parameters that describe it satisfy a particular inequality. By testing which random parameter sets satisfy these inequalities, I show that bistability is necessary to form the segment polarity pattern and serves as a strong predictor of which parameter sets will succeed in forming the pattern. Although the original model was robust to parameter variation, it could not reproduce the observed effects of cell division on the pattern of gene expression. I present a modified version that incorporates recent experimental evidence and does successfully mimic the consequences of cell division. The behavior of this modified model can also be understood in terms of bistability in positive feedback of gene expression. I discuss how this topological property of networks provides robust pattern formation and how large changes in parameters can change the specific pattern produced by a network

Integrating systems biology data to yield functional genomics insights

A report of the recent EMBO Conference 'From Functional Genomics to Systems Biology' held at the EMBL Advanced Training Centre, Heidelberg, Germany, 13-16 November 2010

Cell-fate determination by ubiquitin-dependent regulation of translation.

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination