Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors

Nonsense-mediated decay is well known by the lucid definition of being a RNA surveillance mechanism that ensures the speedy degradation of mRNAs containing premature translation termination codons. However, as we review here, NMD is far from being a simple quality control mechanism; it also regulates the stability of many wild-type transcripts. We summarise the abundance of research that has characterised each of the NMD factors and present a unified model for the recognition of NMD substrates. The contentious issue of how and where NMD occurs is also discussed, particularly with regard to P-bodies and SMG6-driven endonucleolytic degradation. In recent years, the discovery of additional functions played by several of the NMD factors has further complicated the picture. Therefore, we also review the reported roles of UPF1, SMG1 and SMG6 in other cellular processe

tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans

Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNASec however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNASec transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNASec is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNASec genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNASec outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNASec, in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNASec gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatid

Construction of a Suite of Computable Biological Network Models Focused on Mucociliary Clearance in the Respiratory Tract

Mucociliary clearance (MCC), considered as a collaboration of mucus secreted from goblet cells, the airway surface liquid layer, and the beating of cilia of ciliated cells, is the airways’ defense system against airborne contaminants. Because the process is well described at the molecular level, we gathered the available information into a suite of comprehensive causal biological network (CBN) models. The suite consists of three independent models that represent (1) cilium assembly, (2) ciliary beating, and (3) goblet cell hyperplasia/metaplasia and that were built in the Biological Expression Language, which is both human-readable and computable. The network analysis of highly connected nodes and pathways demonstrated that the relevant biology was captured in the MCC models. We also show the scoring of transcriptomic data onto these network models and demonstrate that the models capture the perturbation in each dataset accurately. This work is a continuation of our approach to use computational biological network models and mathematical algorithms that allow for the interpretation of high-throughput molecular datasets in the context of known biology. The MCC network model suite can be a valuable tool in personalized medicine to further understand heterogeneity and individual drug responses in complex respiratory diseases

Report of the 1st and 2nd Mystery of Reactive Oxygen Species Conferences

Non peer reviewe

tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans

Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNASec however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNASec transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNASec is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNASec genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNASec outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNASec, in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNASec gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatids

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of ‘yeast cadmium factor’ were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in our study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification

Genome-wide screens to identify novel components of the metal response in Drosophila melanogaster

Alle Organismen müssen Metallhomöostase sicherstellen, indem sie fluktuierende Mengen von Spurenelementen in der Umwelt bewältigen. Genetische und biochemische Studien in Säuger und Fliegen zeigten eine zentrale Rolle für den metalloregulatorische Protein MTF-1 (metal-responsive transcription factor) in der Homöostase von Übergangsmetallen. MTF-1 ist ein essentielles Gen in Säugern, währenddem es in Drosophila nicht überlebenswichtig ist. In beiden Fällen aber, in der Fliege und in Säugern, ist es ein kritischer Faktor für die Toleranz gegenüber Metallen und in Drosophila, unerwarteterweise auch gegenüber Kupfermangel. Obwohl grosse Fortschritte in der Biologie der Übergangsmetallen gemacht worden sind, bleibt das Wissen über Signalwege und Mechanismen der Antwort auf Metallstress lückenhaft. Auch ist wenig bekannt über die Gene, die in der unmittelbaren Handhabung von verschiedenen Metallen involviert sind. In dieser Studie versuchten wir, neue Komponenten der Antwort auf Metallstress in Drosophila melanogaster mit Hilfe einer Microarray basierten Transkriptom Analyse und genomweiter RNA Interferenz zu identifizieren. Zuerst wurde das Transkriptom von MTF-1 mutanten und wildtyp Larven verglichen. Beide Genotypen wurden in normalem Futter und solchem mit Metallzusätzen aufgezogen. Dadurch wurden neue, mögliche MTF-1 Zielgene gefunden, wie z.B. der Kupferimporter Ctr1B, der Zinkexporter ZnT-D1, der ABC Transporter CG10505 und die Ferritin-Gene Fer1HCH und Fer2LCH. Weitere biochemische und genetische Analysen in der Fliege bestätigten diese Kandidaten als MTF-1 Zielgene und erklärten einige Aspekte des Drosophila MTF-1 knockout Phänotyps. Ausserdem entdeckten wir eine Anzahl von Genen, die unabhängig von MTF-1 auf Metallzusatz reagierten. Unter diesen sind die Gene des Glutathion basierten Detoxifikation Signalwegs und die Gene involviert in Hitzeschock und Immunantwort. In der zweiten Analyse wurde ein genomweiter RNAi Screen in Drosophila S2 Zellkulturen durchgeführt, in Zusammenarbeit mit dem RNAi Screening Center an der Harvard Medical School. Der Screen identifizierte eine grosse Anzahl von Kandidatengenen, welche den Promotor vom Metallothionein A (MtnA) regulieren. MtnA ist eines der am Besten charakterisierten Zielgenen von MTF-1. Ein zweiter Screen mit hausinternen RNAi Verfahren wurde durchgeführt, um die Daten zu bestätigen. Einige Kandidatengene scheinen in einen Signalweg involviert zu sein, der den MtnA promotor reguliert. Weitere Studien sind notwendig für eine detaillierte Charakterisierung dieser Gene, und um ihren Platz in der Hierarchie der Antwort auf Metallstress zu bestimmen. All organisms have to ensure metal homeostasis by coping with fluctuating amounts of trace elements in the environment. Genetic and biochemical studies in mammals and Drosophila have established a central role for metal-responsive transcription factor MTF-1 in transition metal homeostasis. MTF-1 is an essential gene in mammals. In Drosophila, MTF-1 is not required for viability. However, in the fly, as in mammals, it is a critical factor for metal tolerance, and, unexpectedly, also for the copper starvation response. Even though great progress in the biology of transition metals has been made, there is still a lack of knowledge about the pathways and mechanisms leading to metal response and the genes involved in the immediate handling of various metals. In this study we attempted to identify novel components of the metal response using microarray based transcriptome analysis and genome-wide RNA interference technique in Drosophila melanogaster. Firstly, the transcriptome of MTF-1 mutant and wild type larvae, raised in normal or metal-supplemented food, was compared. This revealed new candidate MTF-1 target genes, such as the copper importer Ctr1B, the zinc exporter ZnT-D1, the ABC transporter CG10505, and the ferritin genes Fer1HCH and Fer2LCH. Further biochemical and genetic analyses in the fly established these candidates as genuine MTF-1 regulated genes and explained various aspects of Drosophila MTF-1 knockout phenotype. Moreover, we have uncovered a variety of genes that respond to metal load, independent of MTF-1. Among these are the genes involved in the glutathione- mediated detoxification pathway, and the genes of heat shock and immune responses. In the second analysis a genome-wide RNAi screen was done in cultured Drosophila S2 cells in collaboration with the RNAi Screening Center at Harvard Medical School. The screen identified a large number of candidate genes acting upstream of the promoter of metallothionein A, one of the best characterized target genes of MTF-1. A secondary screen using in house RNAi procedures was performed to validate the data. Several candidate genes appear to be involved in a pathway that regulates the metallothionein promoter. Future studies are needed for a detailed characterization of these genes and for placing them within the hierarchy of the metal stress response pathway