Tõnu Reintamm

Evolution of the 2′-5′-Oligoadenylate Synthetase Family in Eukaryotes and Bacteria

The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins in bacteria that contains the five OAS-specific motifs. This indicates a specific relationship to OAS. The wide distribution of the OAS genes has made it possible to suggest how the OAS1 gene could have evolved from a common ancestor to choanoflagellates and metazoans. Furthermore, we suggest that the OASL may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS may differ from its function in the interferon system, and that this original function of OAS is preserved even in OAS genes that code for proteins, which do not have 2′-5′-oligoadenylate synthetase activity.

Natural occurrence of 2',5'-linked heteronucleotides in marine sponges.

2′,5′-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2′,5′-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2′-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2′,5′-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2′,5′-linked heteronucleotides which accompanied high levels of 2′,5′-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2′p5′G, A2′ p5′U, A2′p5′C, G2′p5′A and G2′ p5′U. The natural occurrence of 2′-adenylated NAD+ was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2′,5′-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family.

Sponge OAS has a distinct genomic structure within the 2-5A synthetase family

Abstract2′,5′-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure. The genomic structure of vertebrate OASs has been thoroughly examined, making it possible to elucidate molecular evolution and expansion of this gene family. Until now, no OAS gene structure was available from sponges to compare it with the corresponding genes from higher organisms. In the present work, we determined the exon/intron structure of the OAS gene from the marine sponge Geodia cydonium and found it to be completely different from the strictly conserved exon/intron pattern of the OAS genes from vertebrates. This finding was corroborated by the analysis of OAS genes from another sponge, Amphimedon queenslandica, whose genome was recently sequenced. Our data suggest that vertebrate and sponge OAS genes have no direct common intron-containing ancestor and two (sub)types of OAS may be discriminated. This study opens new perspectives for understanding the phylogenesis and evolution of 2-5A synthetases as well as functional aspects of this multigene family.

Molecular identification, characterization and distribution of freshwater sponges (Porifera: Spongillidae) in Estonia

This study is the first comprehensive survey of the distribution of freshwater sponges in the rivers of Estonia. The classical morphological method for the identification of freshwater sponges was complemented by a molecular approach, based on sequencing of a DNA region containing a variable D3 domain of the 28S rDNA. This particular segment of 28S RNA proved to be suitable for the discrimination between freshwater sponges at the species level. We showed that Ephydatia fluviatilis was the most widely distributed species followed by Spongilla lacustris and Ephydatia muelleri. Eunapius fragilis was found in a small number of localities and has been described for the first time in Estonia.

Intragenomic Profiling Using Multicopy Genes: The rDNA Internal Transcribed Spacer Sequences of the Freshwater Sponge Ephydatia fluviatilis.

Multicopy genes, like ribosomal RNA genes (rDNA), are widely used to describe and distinguish individuals. Despite concerted evolution that homogenizes a large number of rDNA gene copies, the presence of different gene variants within a genome has been reported. Characterization of an organism by defining every single variant of tens to thousands of rDNA repeat units present in a eukaryotic genome would be quite unreasonable. Here we provide an alternative approach for the characterization of a set of internal transcribed spacer sequences found within every rDNA repeat unit by implementing direct sequencing methodology. The prominent allelic variants and their relative amounts characterizing an individual can be described by a single sequencing electropherogram of the mixed amplicon containing the variants present within the genome. We propose a method for rational analysis of heterogeneity of multicopy genes by compiling a profile based on quantification of different sequence variants of the internal transcribed spacers of the freshwater sponge Ephydatia fluviatilis as an example. In addition to using conventional substitution analysis, we have developed a mathematical method, the proportion model method, to quantify the relative amounts of allelic variants of different length using data from direct sequencing of the heterogeneous amplicon. This method is based on determining the expected signal intensity values (corresponding to peak heights from the sequencing electropherogram) by sequencing clones from the same or highly similar amplicon and comparing hypothesized combinations against the values obtained by direct sequencing of the heterogeneous amplicon. This method allowed to differentiate between all specimens analysed.

RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications.

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2’-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3°C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.

Evolutionary distribution of deoxynucleoside 5-monophosphate N-glycosidase, DNPH1

Deoxynucleoside 5-monophosphate N-glycosidase, DNPH1 is a member of the nucleoside 2-deoxyribosyltransferase (NDT) family. This enzyme catalyzes the hydrolysis of deoxynucleoside monophosphates into free nucleobase moieties and 2-deoxyribose 5-phosphates. The DNPH1 enzymatic activity was first demonstrated in rats and then in humans. Subsequently the DNPH1 gene was identified in a variety of organisms, mainly in Metazoa. Herein, we demonstrate that despite DNPH1 genes being distributed in almost all metazoans, the occurrence of DNPH1genes is mosaic. For example, they cannot be found anywhere in the entire clade of Sauropsida or anywhere in the whole phyla of Arthropoda and Ctenophora. Even among mammals, there are organisms without functional DNPH1 protein (Camelidae and most likely Cetacea). By our knowledge, the DNPH1 gene is missing in plants, fungi and in majority of protists. Accordingly, the enzyme is apparently not of vital importance in all the branches of the Tree of Life. Surprisingly the DNPH1 gene may be found in archaea as well as in bacteria. This refers to the origin of the gene from the period before the archaea branched off from other bacteria. We show that the genomic and protein primary structures of DNPH1 are highly conserved and any modification in such a structure would result in conversion to a pseudogene, which could possibly be eliminated from the genome.