Conservation of the RNA Transport Machineries and Their Coupling to Translation Control across Eukaryotes

Restriction of proteins to discrete subcellular regions is a common mechanism to establish cellular asymmetries and depends on a coordinated program of mRNA localization and translation control. Many processes from the budding of a yeast to the establishment of metazoan embryonic axes and the migration of human neurons, depend on this type of cell polarization. How factors controlling transport and translation assemble to regulate at the same time the movement and translation of transported mRNAs, and whether these mechanisms are conserved across kingdoms is not yet entirely understood. In this review we will focus on some of the best characterized examples of mRNA transport machineries, the “yeast locasome” as an example of RNA transport and translation control in unicellular eukaryotes, and on the Drosophila Bic-D/Egl/Dyn RNA localization machinery as an example of RNA transport in higher eukaryotes. This focus is motivated by the relatively advanced knowledge about the proteins that connect the localizing mRNAs to the transport motors and the many well studied proteins involved in translational control of specific transcripts that are moved by these machineries. We will also discuss whether the core of these RNA transport machineries and factors regulating mRNA localization and translation are conserved across eukaryotes

On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer

Drosophila Xpd Regulates Cdk7 Localization, Mitotic Kinase Activity, Spindle Dynamics, and Chromosome Segregation

The trimeric CAK complex functions in cell cycle control by phosphorylating and activating Cdks while TFIIH-linked CAK functions in transcription. CAK also associates into a tetramer with Xpd, and our analysis of young Drosophila embryos that do not require transcription now suggests a cell cycle function for this interaction. xpd is essential for the coordination and rapid progression of the mitotic divisions during the late nuclear division cycles. Lack of Xpd also causes defects in the dynamics of the mitotic spindle and chromosomal instability as seen in the failure to segregate chromosomes properly during ana- and telophase. These defects appear to be also nucleotide excision repair (NER)–independent. In the absence of Xpd, misrouted spindle microtubules attach to chromosomes of neighboring mitotic figures, removing them from their normal location and causing multipolar spindles and aneuploidy. Lack of Xpd also causes changes in the dynamics of subcellular and temporal distribution of the CAK component Cdk7 and local mitotic kinase activity. xpd thus functions normally to re-localize Cdk7(CAK) to different subcellular compartments, apparently removing it from its cell cycle substrate, the mitotic Cdk. This work proves that the multitask protein Xpd also plays an essential role in cell cycle regulation that appears to be independent of transcription or NER. Xpd dynamically localizes Cdk7/CAK to and away from subcellular substrates, thereby controlling local mitotic kinase activity. Possibly through this activity, xpd controls spindle dynamics and chromosome segregation in our model system. This novel role of xpd should also lead to new insights into the understanding of the neurological and cancer aspects of the human XPD disease phenotypes

α-Phenylalanyl tRNA synthetase competes with Notch signaling through its N-terminal domain

The alpha subunit of the cytoplasmic Phenylalanyl tRNA synthetase (α-PheRS, FARSA in humans) displays cell growth and proliferation activities and its elevated levels can induce cell fate changes and tumor-like phenotypes that are neither dependent on the canonical function of charging tRNAPhe with phenylalanine nor on stimulating general translation. In intestinal stem cells of Drosophila midguts, α-PheRS levels are naturally slightly elevated and human FARSA mRNA levels are elevated in multiple cancers. In the Drosophila midgut model, elevated α-PheRS levels caused the accumulation of many additional proliferating cells resembling intestinal stem cells (ISCs) and enteroblasts (EBs). This phenotype partially resembles the tumor-like phenotype described as Notch RNAi phenotype for the same cells. Genetic interactions between α-PheRS and Notch suggest that their activities neutralize each other and that elevated α-PheRS levels attenuate Notch signaling when Notch induces differentiation into enterocytes, type II neuroblast stem cell proliferation, or transcription of a Notch reporter. These non-canonical functions all map to the N-terminal part of α-PheRS which accumulates naturally in the intestine. This truncated version of α-PheRS (α-S) also localizes to nuclei and displays weak sequence similarity to the Notch intracellular domain (NICD), suggesting that α-S might compete with the NICD for binding to a common target. Supporting this hypothesis, the tryptophan (W) residue reported to be key for the interaction between the NICD and the Su(H) BTD domain is not only conserved in α-PheRS and α-S, but also essential for attenuating Notch signaling

The aminoacyl-tRNA synthetases of Drosophila melanogaster.

Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS genes, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translational fidelity. However, the lack of a systematic annotation of this gene family has hampered such studies. Here, we report the identification of the entire set of aaRS genes in the fly genome and we predict their roles based on experimental evidence and/or orthology. Further, we propose a new, systematic and logical nomenclature for aaRSs. We also review the research conducted on Drosophila aaRSs to date. Together, our work provides the foundation for further research in the fly aaRS field.S.J.M. is supported by the FlyBase NIH/NHGRI grant U41HG000739 (W.M. Gelbart, Harvard University, PI; N.H. Brown, University of Cambridge, coPI). J.L. was supported by Cancer Research Switzerland and a SNF grant to B.S., and by a SNSF Early Postdoc Fellowship.This is the final version of the article. It was first available from Taylor & Francis via http://dx.doi.org/10.1080/19336934.2015.110119

Systematic Functional Analysis of BicD Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD

Protein phosphorylation is involved in posttranslational control of essentially all biological processes. Using mass spectrometry, recent analyses of whole phosphoproteomes led to the identification of numerous new phosphorylation sites. However, the function of most of these sites remained unknown. We chose the Drosophila Bicaudal-D protein to estimate the importance of individual phosphorylation events. Being involved in different cellular processes, BicD is required for oocyte determination, for RNA transport during oogenesis and embryogenesis, and for photoreceptor nuclei migration in the developing eye. The numerous roles of BicD and the available evidence for functional importance of BicD phosphorylation led us to identify eight phosphorylation sites of BicD, and we tested a total of 14 identified and suspected phosphoserine residues for their functional importance in vivo in flies. Surprisingly, all these serines turned out to be dispensable for providing sufficient basal BicD activity for normal growth and development. However, in a genetically sensitized background where the BicD(A40V) protein variant provides only partial activity, serine 103 substitutions are not neutral anymore, but show surprising differences. The S103D substitution completely inactivates the protein, whereas S103A behaves neutral, and the S103F substitution, isolated in a genetic screen, restores BicD(A40V) function. Our results suggest that many BicD phosphorylation events may either be fortuitous or play a modulating function as shown for Ser(103). Remarkably, amongst the Drosophila serines we found phosphorylated, Ser(103) is the only one that is fully conserved in mammalian BicD

Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development.

Aminoacyl-tRNA synthetases (aaRSs) constitute a family of ubiquitously expressed essential enzymes that ligate amino acids to their cognate tRNAs for protein synthesis. Recently, aaRS mutations have been linked to various human diseases; however, how these mutations lead to diseases has remained unclear. In order to address the importance of aminoacylation fidelity in multicellular organisms, we generated an amino-acid double-sieving model in Drosophila melanogaster using phenylalanyl-tRNA synthetase (PheRS). Double-sieving-defective mutations dramatically misacylate non-cognate Tyr, induce protein mistranslation and cause endoplasmic reticulum stress in flies. Mutant adults exhibit many defects, including loss of neuronal cells, impaired locomotive performance, shortened lifespan and smaller organ size. At the cellular level, the mutations reduce cell proliferation and promote cell death. Our results also reveal the particular importance of the first amino-acid recognition sieve. Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases

Validation of automated artificial intelligence segmentation of optical coherence tomography images

PURPOSE To benchmark the human and machine performance of spectral-domain (SD) and swept-source (SS) optical coherence tomography (OCT) image segmentation, i.e., pixel-wise classification, for the compartments vitreous, retina, choroid, sclera. METHODS A convolutional neural network (CNN) was trained on OCT B-scan images annotated by a senior ground truth expert retina specialist to segment the posterior eye compartments. Independent benchmark data sets (30 SDOCT and 30 SSOCT) were manually segmented by three classes of graders with varying levels of ophthalmic proficiencies. Nine graders contributed to benchmark an additional 60 images in three consecutive runs. Inter-human and intra-human class agreement was measured and compared to the CNN results. RESULTS The CNN training data consisted of a total of 6210 manually segmented images derived from 2070 B-scans (1046 SDOCT and 1024 SSOCT; 630 C-Scans). The CNN segmentation revealed a high agreement with all grader groups. For all compartments and groups, the mean Intersection over Union (IOU) score of CNN compartmentalization versus group graders' compartmentalization was higher than the mean score for intra-grader group comparison. CONCLUSION The proposed deep learning segmentation algorithm (CNN) for automated eye compartment segmentation in OCT B-scans (SDOCT and SSOCT) is on par with manual segmentations by human graders

Systematic Functional Analysis of Bicaudal-D Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD

Protein phosphorylation is involved in posttranslational control of essentially all biological processes. Using mass spectrometry, recent analyses of whole phosphoproteomes led to the identification of numerous new phosphorylation sites. However, the function of most of these sites remained unknown. We chose the Drosophila Bicaudal-D protein to estimate the importance of individual phosphorylation events. Being involved in different cellular processes, BicD is required for oocyte determination, for RNA transport during oogenesis and embryogenesis, and for photoreceptor nuclei migration in the developing eye. The numerous roles of BicD and the available evidence for functional importance of BicD phosphorylation led us to identify eight phosphorylation sites of BicD, and we tested a total of 14 identified and suspected phosphoserine residues for their functional importance in vivo in flies. Surprisingly, all these serines turned out to be dispensable for providing sufficient basal BicD activity for normal growth and development. However, in a genetically sensitized background where the BicDA40V protein variant provides only partial activity, serine 103 substitutions are not neutral anymore, but show surprising differences. The S103D substitution completely inactivates the protein, whereas S103A behaves neutral, and the S103F substitution, isolated in a genetic screen, restores BicDA40V function. Our results suggest that many BicD phosphorylation events may either be fortuitous or play a modulating function as shown for Ser103. Remarkably, amongst the Drosophila serines we found phosphorylated, Ser103 is the only one that is fully conserved in mammalian BicD

Pyrrolidine dithiocarbamate administered during ex-vivo lung perfusion promotes rehabilitation of injured donor rat lungs obtained after prolonged warm ischemia.

Damaged lung grafts obtained after circulatory death (DCD lungs) and warm ischemia may be at high risk of reperfusion injury after transplantation. Such lungs could be pharmacologically reconditioned using ex-vivo lung perfusion (EVLP). Since acute inflammation related to the activation of nuclear factor kappaB (NF-κB) is instrumental in lung reperfusion injury, we hypothesized that DCD lungs might be treated during EVLP by pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. Rat lungs exposed to 1h warm ischemia and 2 h cold ischemia were subjected to EVLP during 4h, in absence (CTRL group, N = 6) or in presence of PDTC (2.5g/L, PDTC group, N = 6). Static pulmonary compliance (SPC), peak airway pressure (PAWP), pulmonary vascular resistance (PVR), and oxygenation capacity were determined during EVLP. After EVLP, we measured the weight gain of the heart-lung block (edema), and the concentration of LDH (cell damage), proteins (permeability edema) and of the cytokines IL-6, TNF-α and CINC-1 in bronchoalveolar lavage (BAL), and we evaluated NF-κB activation by the degree of phosphorylation and degradation of its inhibitor IκBα in lung tissue. In CTRL, we found significant NF-κB activation, lung edema, and a massive release of LDH, proteins and cytokines. SPC significantly decreased, PAWP and PVR increased, while oxygenation tended to decrease. Treatment with PDTC during EVLP inhibited NF-κB activation, did not influence LDH release, but markedly reduced lung edema and protein concentration in BAL, suppressed TNFα and IL-6 release, and abrogated the changes in SPC, PAWP and PVR, with unchanged oxygenation. In conclusion, suppression of innate immune activation during EVLP using the NF-κB inhibitor PDTC promotes significant improvement of damaged rat DCD lungs. Future studies will determine if such rehabilitated lungs are suitable for in vivo transplantation