The importance of proteins of the RNase II/RNB-family in pathogenic bacteria

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Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a "SUPER-ENZYME"

RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3'-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different single- or double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme."The work was supported by Ministerio de Educación y Ciencia, Spain, Grant SAF2007-61926, an institutional grant from the “Fundación Ramón Areces”, and by Fundaçao para a Ciência e a Tecnologia, PortugalS

Iron age genomic data from Althiburos – Tunisia renew the debate on the origins of African taurine cattle

The Maghreb is a key region for understanding the dynamics of cattle dispersal and admixture with local aurochs following their earliest domestication in the Fertile Crescent more than 10,000 years ago. Here, we present data on autosomal genomes and mitogenomes obtained for four archaeological specimens of Iron Age (∼2,800 cal BP–2,000 cal BP) domestic cattle from the Eastern Maghreb, i.e. Althiburos (El Kef, Tunisia). D-loop sequences were obtained for an additional eight cattle specimens from this site. Maternal lineages were assigned to the elusive R and ubiquitous African-T1 haplogroups found in two and ten Althiburos specimens, respectively. Our results can be explained by post-domestication hybridization of Althiburos cattle with local aurochs. However, we cannot rule out an independent domestication in North Africa considering the shared ancestry of Althiburos cattle with the pre-domestic Moroccan aurochs and present-day African taurine cattle.info:eu-repo/semantics/publishedVersio

Dis3L2 regulates cell proliferation and tissue growth though a conserved mechanism

Dis3L2 is a highly conserved 3’-5’ exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms’ tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth

Efficient second harmonic generation by para-nitroaniline embedded in electro-spun polymeric nanofibres

Intense well polarized second harmonic light was generated by poly(methyl methacrylate) nanofibres with embedded para-nitroaniline nanocrystals. Subwavelength diameter fibres were electro-spun using a 1.2 weight ratio of chromophore to polymer. Analysis of the generated second harmonic light indicates that the para-nitroaniline molecules, which nominally crystalize in the centrosymmetric space group, were organized into noncentrosymmetric structures leading to a second order susceptibility dominated by a single tensor element. Under the best deposition conditions, the nanofibrers display an effective nonlinear optical susceptibility approximately two orders of magnitude greater than that of potassium dihydrogen phosphate. Generalizing this approach to a broad range of organic molecules with strong individual molecular second order nonlinear responses, but which nominally form centrosymmetric organic crystals, could open a new pathway for the fabrication of efficient sub-micron sized second harmonic light generators.Hugo Goncalves thanks the Portuguese Foundation for Science and Technology (FCT) for the support under grant PD/BD/111873/2015. The equipment used to characterize the second harmonic response of the electro-spun fibres was acquired within the framework of the Portuguese National Program for Scientific Re-equipment, contract REEQ-25/FIS/2005 with funds from POCI 2010 (FEDER) and FCT. The Raman microscope was acquired through the project nSTeP Nanostructured Systems for Tailored Performance, NORTE-07-0124-FEDER-000039, ON. 2. This work was in part developed in the scope of the projects CICECO-Aveiro Institute of Materials (UID/CTM/50011/2013), financed by national funds through the Fundacao para a Ciencia e a Tecnologia/Ministerio da Educacao e Ciencia (FCT/MEC) and co-financed by FEDER under the PT2020 Partnership Agreement. The authors are grateful to A M P Botas (University of Aveiro) for help in acquiring the hyperspectral microscopy data.info:eu-repo/semantics/acceptedVersio

Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress

The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5′-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3′. Using surface plasmon resonance experiments and electrophoretic mobility shift assays it is demonstrated that Haa1 interacts with high affinity (KD of 2 nM) with the HRE motif present in the ACRE region of TPO3 promoter. No significant interaction was found between Haa1 and HRE motifs having adenine nucleotides at positions 6 and 8 (KD of 396 and 6780 nM, respectively) suggesting that Haa1p does not recognize these motifs in vivo. A lower affinity of Haa1 toward HRE motifs having mutations in the guanine nucleotides at position 7 and 9 (KD of 21 and 119 nM, respectively) was also observed. Altogether, the results obtained indicate that the minimal functional binding site of Haa1 is 5′-(G/C)(A/C)GG(G/C)G-3′. The Haa1-dependent transcriptional regulatory network active in yeast response to acetic acid stress is proposed

DIS3 isoforms vary in their endoribonuclease activity and are differentially expressed within haematological cancers

DIS3 is the catalytic subunit of the exosome, a protein complex involved in the 3’ to 5’ degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two proteincoding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino-acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 as well as the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared to isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared to isoform 2 whereas Acute Myeloid Leukemia (AML) and chronic myelomonocytic leukaemia (CMML) patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers

Biochemical characterization of campylobacter jejuni pnpase, an exoribonuclease important for bacterial pathogenicity

International audienceBacteria need to promptly respond to environmental changes. Ribonucleases (RNases) are key factors in the adaptation to new environments by enabling a rapid adjustment in RNA levels. The exoribonuclease polynucleotide phosphorylase (PNPase) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization in C. jejuni. However, the mechanism of action of this ribonuclease is not yet known. In this work we have characterized the biochemical activity of C. jejuni PNPase. Our results demonstrate that Cj-PNP is a processive 3′ to 5′ exoribonuclease that degrades single-stranded RNAs. Its activity is regulated according to the temperature and divalent ions. We have also shown that the KH and S1 domains are important for trimerization, RNA binding, and, consequently, for the activity of Cj-PNP. These findings will be helpful to develop new strategies for fighting against C. jejuni and may be extrapolated to other foodborne pathogens