Role of RNA editing in RNA splicing and in nuclear export of microRNA precursors

Doppelstrang RNA-bindende Domänen (dsRBDs) sind häufige Motive in Proteinen die in RNA Prozessierung, Lokalisierung oder RNA Interferenz involviert sind. Für RNA Bindung spielt dabei die Sekundärstruktur der RNA und nicht ihre Sequenz die entscheidende Rolle. „Adenosine deaminases that act on RNA” (ADARs) sind dsRBD-beinhaltende Proteine, deren Aufgabe die Deaminierung von Adenosin zu Inosin in spezifischen doppelsträngigen RNA Substraten ist. Die modifizierten Basen befinden sich häufig nahe an 5´ Spleißstellen und haben infolgedessen einen breiten Einfluß auf RNA Spleißen und Stabilität. Korrektes Spleißen der mRNA der Untereinheit B des Glutamatrezeptors benötigt zum Beispiel RNA Editierung durch ADAR an mehreren Stellen, unter anderem auch an der R/G Position in Exon 13, die nur 2 Nukleotide von der 5´ Spleißstelle liegt. Der genaue Mechanismus, wie Deaminierung an der R/G Position Spleißen beeinflußt, ist jedoch unklar. Wir vermuteten, dass Inosine an dieser kritischen Position die Interaktion der 5´ Spleißstelle mit U1 snRNA ändert. Diese Hypothese wurde experimentell durch eine kompensierende U1 snRNA Mutante getestet. Weiters weist die Umgebung der R/G Stelle Ähnlichkeiten mit spleißhemmenden Sequenzen auf, an denen reprimierende Proteine wie hnRNP A1 und hnRNP H binden. Deswegen wurde auch die Interaktion dieser Faktoren mit der R/G Stelle untersucht. Die Vorläufer der microRNAs („microRNA precursors“) stellen eine weitere bedeutende Klasse von ADAR Substraten dar. Die Prozessierung dieser Vorläufer ist ebenfalls stark durch RNA Editierung geändert. Sie werden, zum Beispiel, suszeptibel für den Abbau durch die cytoplasmatische Ribonuklease Tudor-SN. Da ADAR1 ein dsRNA-bindendes Protein ist, das zwischen Nukleus und Cytoplasma wandert und mit Exportin5, dem Hauptexportrezeptor der microRNA Vorläufer interagiert, wurden co-immunpräzipitierende Methoden angewandt, um die Interaktion von ADAR1 und editierten microRNA Vorläufer sowohl im Nukleus als auch im Cytoplasma zu untersuchen. In eukaryotischen Genomen findet man „Short Interspersed Elements“ (SINEs) als transposable Elemente, die durch ein RNA Intermediat mobilisiert werden. Dieses RNA Intermediat interagiert mit dsRBD-beinhaltenden Proteinen, die wichtig für Transkription und Translation sind. Diesbezüglich wurde gezeigt, dass pflanzliches SB1 SINE RNA mit dem an partielle doppelsträngige RNA bindenden Protein HYL1, jedoch nicht mit DRB4, das für die perfekt doppelsträngige RNA Struktur spezifisch ist, in vitro interagiert. HYL1 ist ein wichtiger Faktor der microRNA und tasiRNA Produktion in Pflanzen. Die Bindestelle für HYL1 an SINE RNA befindet sich an einer „stem-loop“ Struktur, die stark an microRNA Vorläufer erinnert. Dies lässt Schlüsse zu, wie SINE RNAs RNAi Wege bestimmen könnten.The double-stranded RNA binding domain (dsRBD) is a common motif found in proteins involved in RNA processing, localisation or interference. RNA recognition by these proteins depends on the secondary structure and not on the sequence. Adenosine deaminases that act on RNA (ADARs) are dsRBD-containing proteins which modify target adenosines into inosines in double-stranded RNA substrates, frequently in vicinity of 5´ splice sites, having a profound impact on the subsequent RNA processing events, including RNA splicing and stability. Accurate splicing of messenger RNAs coding for the subunit B of the glutamate receptor, for example, relies upon ADAR-mediated RNA editing at several positions, including the R/G site located in exon 13, just two nucleotides upstream of the exon-intron border. The exact macromolecular interaction affected by deamination at this site, however, has not been identified. We postulated that inosine in this critical position influences U1 snRNA base-pairing to this imperfect 5´ splice site. This hypothesis was tested by employing compensatory U1 snRNA mutants. Furthermore, the sequence surrounding the edited R/G site strongly resembles a splicing silencer consensus bound by splicing repressor proteins, like hnRNP A1 or hnRNP H. Therefore, the interaction between R/G site and these proteins was also investigated. MicroRNA precursors represent another prominent class of ADAR substrates whose processing is substantially altered upon RNA editing, as they become sensitive to cleavage by Tudor-SN, a cytoplasmic ribonuclease. Since ADAR1 is a dsRNA-binding shuttling protein that interacts with Exportin5, a main export receptor for miRNA precursors, a co-immunoprecipitation method was exerted to detect the interaction of ADAR1 and edited miRNA precursors in both nucleus and cytoplasm. In eukaryotic genomes, short interspersed elements (SINEs) are transposable regions mobilised through an RNA intermediate that interacts with dsRBD-containing proteins crucial for transcription and translation control. In this part of the study, it is being shown that plant SB1 SINE RNA interacts strongly with imperfect dsRNA-binding protein HYL1, a key factor in the microRNA and trans-acting small interfering RNA (tasiRNA) production in plants, but not with perfect dsRNA-specific protein DRB4 in vitro. The binding site maps to stem-loop structure highly reminiscent of miRNA precursors, suggesting how SINE RNAs could regulate different RNAi pathways

Seril-tRNA-sintetaze iz metanogenih arheja: supresija bakterijskih amber mutacija i heterologna toksičnost

Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation.Metanogene arheje imaju neobične seril-tRNA-sintetaze (SerRS), evolucijski udaljene od SerRS koje se mogu naći u drugih arheja, eukariota i bakterija. Naša nedavna analiza kristalne strukture SerRS iz metanogene arheje Methanosarina barkeri otkrila je karakterističnu N-terminalnu domenu i katalitički ion cinka na aktivnom mjestu. Da bi se rasvijetlio način na koji metanogeni tip SerRS diskriminira supstrate, autori su istraživali in vivo interakciju metanogenog tipa SerRS s pripadnim molekulama tRNA u bakteriji Escherichia coli ili kvascu Saccharomyces cerevisiae. Ekspresija raznih SerRS metanogenoga tipa bila je toksična za bakteriju E. coli, te je rezultirala sintezom proteina s greškama u aminokiselinskom sastavu, što se vidi iz testa stabilnosti β-galaktozidaze. Iako je SerRS iz metanogenih arheja mogla prepoznati tRNASer iz sve tri domene života in vitro, vjerojatno je toksičnost zasjenila komplementaciju funkcije endogene SerRS u bakteriji E. coli i kvascu S. cerevisiae. Međutim, unatoč toksičnosti, koekspresija metanogenoga tipa SerRS s pripadnom tRNA suprimirala je bakterijsku amber mutaciju

SINE RNA Induces Severe Developmental Defects in Arabidopsis thaliana and Interacts with HYL1 (DRB1), a Key Member of the DCL1 Complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes

Seryl-tRNA Synthetases from Methanogenic Archaea: Suppression of Bacterial Amber Mutation and Heterologous Toxicity

Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation