Structure-function relationships, tertiary interactions and thermostability of RNase P

Identification of rnpB and rnpA in the Aquificales. Since the Aquificales (the close relatives of A. aeolicus) represent a group of bacteria for which not much information about RNase P RNA is available, we wanted to scrutinize if other members of this subphylum beside A. aeolicus also display idiosyncrasies regarding this essential enzyme. In cooperation with a bioinformatic research group (Gerhard Steger, Düsseldorf) we identified the P RNA (rnpB) and protein (rnpA) genes in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. Their P RNAs adhere to the bacterial P RNA consensus and proved to be active catalysts at high metal ion concentrations in vitro and could be activated by heterologous bacterial P proteins at low salt. Both RNAs lack helix P18 and thus one of the three tetraloop-helix interactions that bridge S-and C-domains. S. azorense and P. marina P RNA are more thermostable than E. coli P RNA and require higher temperatures for proper folding. The P protein genes (rnpA) of S. azorense and P. marina were identified as well and co-localize with the rmpH gene encoding ribosomal protein L34 as in the majority of bacteria. We also identified RNase P activity in other Aquificales (Aquifex pyrophilus, Hydrogenobacter thermophilus TK 6 and Thermocrinis ruber) and demonstrated that active RNase P holoenzymes can be reconstituted from their total RNA upon addition of the E. coli or B.subtilis P protein. We were further able to demonstrate that a close relative of A. aeolicus, A. pyrophilus, is also lacks the rnpA gene in the canonical bacterial genomic context. Finally, we succeeded in detecting RNase P activity in fractions of A. aeolicus cell lysates and demonstrated that the enzyme possesses an essential protein component that, unlike in other bacterial RNase P enzymes, cannot be substituted for by E. coli or B. subtilis P proteins. Strucural basis of a ribozyme’s thermostability: P1-L9 interdomain interaction in RNase P RNA. The two independent folding domains of type A bacterial P RNAs are interconnected by three long-range tertiary interaction: P1-L9, L8-P4 and P8-L18. Though predicted by phylogenetic analyses and confirmed by X-ray crystallography, the precise structural and functional role of these interactions is largely unclear. Our analysis of the P RNAs from the thermophilic Aqificales S. azorense and P. marina (see above) revealed that these P RNAs and the one from the thermophile Thermus thermophilus share a 5'-GYAA L9 tetraloop and a P1 receptor site consisting of a G-C bp tandem, a combination not present in other bacteria. Also, helices P1 and P9 were observed to be stabilized in P RNAs from thermophiles by helix extension and/or deletion of nucleotide bulges. These observations prompted us to scrutinize the importance of the P1-L9 interaction in P RNA of the thermophile Thermus thermophilus and to compare it to that of the mesophile E. coli. The P1-L9 contact indeed turned out to be crucial for folding and activity of T. thermophilus P RNA at high temperatures and low magnesium ion concentrations as present in holoenzyme reactions. I showed by native PAGE that the P1-L9 interaction module represents the key anchoring points towards folding into the most active RNA conformer of T. thermophilus P RNA. In contrast, disruption of this interaction in the P RNA from the mesophile E. coli did not abrogate functionality in vivo or in vitro. However, exchanging the P1-L9 module in E. coli P RNA for that of T. thermophilus generated a thermostable E. coli variant. I also replaced the P1-L9 interaction module in E. coli P RNA with an alternative pseudoknot interaction module present in some Mycoplasma P RNAs, which again resulted in thermostabilization of the chimeric P RNA. This work for the first time demonstrates that the module P1-L9 is directly linked to thermostabilization of P RNAs. This finding is an important step towards understanding structure-function relationships of catalytic RNAs

Minor changes largely restore catalytic activity of archaeal RNase P RNA from Methanothermobacter thermoautotrophicus

The increased protein proportion of archaeal and eukaryal ribonuclease (RNase) P holoenzymes parallels a vast decrease in the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus substantially activate the catalytic (C-) domain of archaeal P RNA from Methanothermobacter, in the absence and presence of the bacterial RNase P protein. Large increases in ribozyme activity required the cooperative effect of at least two structural alterations. The P1 helix of P RNA from Methanothermobacter was found to be extended, which increases ribozyme activity (ca 200-fold) and stabilizes the tertiary structure. Activity increases of mutated archaeal C-domain variants were more pronounced in the context of chimeric P RNAs carrying the bacterial specificity (S-) domain of Escherichia coli instead of the archaeal S-domain. This could be explained by the loss of the archaeal S-domain's capacity to support tight and productive substrate binding in the absence of protein cofactors. Our results demonstrate that the catalytic capacity of archaeal P RNAs is close to that of their bacterial counterparts, but is masked by minor changes in the C-domain and, particularly, by poor function of the archaeal S-domain in the absence of archaeal protein cofactors

STAT3 modulates CD4+ T mitochondrial dynamics and function in aging

Aging promotes numerous intracellular changes in T cells that impact their effector function. Our data show that aging promotes an increase in the localization of STAT3 to the mitochondria (mitoSTAT3), which promotes changes in mitochondrial dynamics and function and T-cell cytokine production. Mechanistically, mitoSTAT3 increased the activity of aging T-cell mitochondria by increasing complex II. Limiting mitoSTAT3 using a mitochondria-targeted STAT3 inhibitor, Mtcur-1 lowered complex II activity, prevented age-induced changes in mitochondrial dynamics and function, and reduced Th17 inflammation. Exogenous expression of a constitutively phosphorylated form of STAT3 in T cells from young adults mimicked changes in mitochondrial dynamics and function in T cells from older adults and partially recapitulated aging-related cytokine profiles. Our data show the mechanistic link among mitoSTAT3, mitochondrial dynamics, function, and T-cell cytokine production

The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly

Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates

Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ∼70–85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III

Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs

Struktur-Funktionsanalyse, Tertiärinteraktionen und Thermostabilität von RNase P

Identification of rnpB and rnpA in the Aquificales. Since the Aquificales (the close relatives of A. aeolicus) represent a group of bacteria for which not much information about RNase P RNA is available, we wanted to scrutinize if other members of this subphylum beside A. aeolicus also display idiosyncrasies regarding this essential enzyme. In cooperation with a bioinformatic research group (Gerhard Steger, Düsseldorf) we identified the P RNA (rnpB) and protein (rnpA) genes in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. Their P RNAs adhere to the bacterial P RNA consensus and proved to be active catalysts at high metal ion concentrations in vitro and could be activated by heterologous bacterial P proteins at low salt. Both RNAs lack helix P18 and thus one of the three tetraloop-helix interactions that bridge S-and C-domains. S. azorense and P. marina P RNA are more thermostable than E. coli P RNA and require higher temperatures for proper folding. The P protein genes (rnpA) of S. azorense and P. marina were identified as well and co-localize with the rmpH gene encoding ribosomal protein L34 as in the majority of bacteria. We also identified RNase P activity in other Aquificales (Aquifex pyrophilus, Hydrogenobacter thermophilus TK 6 and Thermocrinis ruber) and demonstrated that active RNase P holoenzymes can be reconstituted from their total RNA upon addition of the E. coli or B.subtilis P protein. We were further able to demonstrate that a close relative of A. aeolicus, A. pyrophilus, is also lacks the rnpA gene in the canonical bacterial genomic context. Finally, we succeeded in detecting RNase P activity in fractions of A. aeolicus cell lysates and demonstrated that the enzyme possesses an essential protein component that, unlike in other bacterial RNase P enzymes, cannot be substituted for by E. coli or B. subtilis P proteins. Strucural basis of a ribozyme’s thermostability: P1-L9 interdomain interaction in RNase P RNA. The two independent folding domains of type A bacterial P RNAs are interconnected by three long-range tertiary interaction: P1-L9, L8-P4 and P8-L18. Though predicted by phylogenetic analyses and confirmed by X-ray crystallography, the precise structural and functional role of these interactions is largely unclear. Our analysis of the P RNAs from the thermophilic Aqificales S. azorense and P. marina (see above) revealed that these P RNAs and the one from the thermophile Thermus thermophilus share a 5'-GYAA L9 tetraloop and a P1 receptor site consisting of a G-C bp tandem, a combination not present in other bacteria. Also, helices P1 and P9 were observed to be stabilized in P RNAs from thermophiles by helix extension and/or deletion of nucleotide bulges. These observations prompted us to scrutinize the importance of the P1-L9 interaction in P RNA of the thermophile Thermus thermophilus and to compare it to that of the mesophile E. coli. The P1-L9 contact indeed turned out to be crucial for folding and activity of T. thermophilus P RNA at high temperatures and low magnesium ion concentrations as present in holoenzyme reactions. I showed by native PAGE that the P1-L9 interaction module represents the key anchoring points towards folding into the most active RNA conformer of T. thermophilus P RNA. In contrast, disruption of this interaction in the P RNA from the mesophile E. coli did not abrogate functionality in vivo or in vitro. However, exchanging the P1-L9 module in E. coli P RNA for that of T. thermophilus generated a thermostable E. coli variant. I also replaced the P1-L9 interaction module in E. coli P RNA with an alternative pseudoknot interaction module present in some Mycoplasma P RNAs, which again resulted in thermostabilization of the chimeric P RNA. This work for the first time demonstrates that the module P1-L9 is directly linked to thermostabilization of P RNAs. This finding is an important step towards understanding structure-function relationships of catalytic RNAs.Identifizierung von rnpB und rnpA in den Aquificales Da die Aquificales (die nächsten Verwandten von Aquifex aeolicus) eine Gruppe von Bakterien darstellen, für die wenig über RNase P bekannt ist, wollten wir untersuchen, ob auch andere Mitglieder dieses Subphylums außer Aquifex aeolicus Besonderheiten hinsichtlich dieses essentiellen Enzyms aufweisen. In Zusammenarbeit mit einer Bioinformatik-Forschungsgruppe (Gerhard Steger, Düsseldorf) identifizierten wir die Gene für die P RNAs (rnpB) und P Proteine (rnpA) der beiden Aquificales Sulfurihydrogenibium azorense und Persephonella marina. Ihre P RNAs entsprechen dem bakteriellen P RNA-Konsensus, erwiesen sich bei hohen Metallionenkonzentrationen als aktive Katalysatoren und konnten unter Niedrigsalzbedingungen durch heterologe bakterielle P Proteine aktiviert werden. Beiden P RNAs fehlt die Helix P18 und damit eine der drei Tetraloop-Helix-Interaktionen, die die S- und die C-Domäne miteinander verbrücken. Die P RNAs von S. azorense und P. marina sind thermostabiler als die P RNA von E. coli und bedürfen zur korrekten Faltung höherer Temperaturen. Die Gene für die P Proteine (rnpA) von S. azorense und P. marina wurden ebenfalls identifiziert und sind, wie in den meisten Bakterien, im Genom mit dem rpmH-Gen, codierend für das ribosomale Protein L34, co-lokalisiert. Weiterhin identifizierten wir RNase P-Aktivität auch in anderen Aquificales (Aquifex pyrophilus, Hydrogenobacter thermophilus TK 6 und Thermocrinis ruber) und zeigten, daß durch Zugabe von P Protein von E. coli oder B. subtilis aus der Total-RNA dieser Aquificales aktive RNase P-Holoenzyme rekonstituiert werden können. Darüber hinaus konnten wir zeigen, daß einem nahen Verwandten von A. aeolicus, A. pyrophilus, das rnpA-Gen im kanonischen Kontext ebenfalls fehlt. Schließlich gelang es uns, RNase P-Aktivität in Fraktionen von A. aeolicus-Zelllysaten zu detektieren und zu zeigen, dass hier das Enzym eine essenzielle Proteinkomponente besitzt, die, anders als bei anderen bakteriellen RNase P-Enzymen, nicht durch E. coli- oder B. subtilis- RNase P Proteine ersetzbar ist. Strukturelle Grundlagen von Ribozym-Stabilität: die P1-L9-Interdomäneninteraktion in RNase P-RNA Die beiden unabhängigen Faltungsdomänen von bakteriellen Typ A-RNase P RNAs sind durch drei weitreichende Tertiärinteraktionen miteinander verbunden: P1-L9, L8-P4 und P8-L18. Obwohl sich diese durch phylogenetische Analysen vorhersagen und durch Röntgenkristallographie bestätigen ließen, ist die genaue strukturelle und funktionelle Bedeutung dieser Interaktionen weitgehend unklar. Unsere Analyse der P RNAs der thermophilen Aquificales S. azorense und P. marina (s. o.) zeigte, daß diese P RNAs, wie auch die des thermophilen Bakteriums Thermus thermophilus, in einem 5’-GYAA L9-Tetraloop sowie einer P1-Rezeptor, bestehend aus einem Tandem-GC-Basenpaar, übereinstimmen – eine Kombination, die man in anderen Bakterien so nicht findet. Darüber hinaus wurde beobachtet, daß Helices P1 und P9 in P RNAs von thermophilen Bakterien durch Helix-Verlängerungen und/oder Deletion von ungepaarten Nukleotiden („Bulges“) stabilisiert werden. Diese Beobachtungen veranlassten uns, die Bedeutung der P1-L9-Interaktion in P RNAs des thermophilen Bakteriums Thermus thermophilus zu untersuchen und mit der in dem mesophilen Bakterium E. coli zu vergleichen. Der P1-L9-Kontakt erwies sich tatsächlich als von fundamentaler Bedeutung für die Faltung und Aktivität der T. thermophilus-RNase P RNA bei hohen Temperaturen sowie bei den niedrigen Magnesiumionenkonzentrationen der Holoenzym-Reaktion. Durch native PAGE zeigte ich, daß die P1-L9-Interaktion ein wesentlicher Ankerpunkt für die Faltung in das aktivste RNA-Konformer der T. thermophilus-RNase P RNA ist. Im Gegensatz dazu bewirkte die Störung dieser Interaktion im mesophilen Bakterium E. coli weder eine Beeinträchtigung der Funktion in vivo noch in vitro. Dagegen führte der Austausch des P1-L9-Moduls von E. coli RNase P RNA gegen das aus T. thermophilus zu einer thermostabilen E. coli-P RNA-Variante. Ich ersetzte das P1-L9-Interaktionsmodul der E. coli-P RNA außerdem durch eine alternative Pseudoknoten-Struktur, die in manchen Mycoplasma-P RNAs an korrepondierender Stelle zu finden ist, was ebenfalls zu einer Thermostabilisierung der chimären P RNA führte. Diese Arbeit zeigt zum ersten Mal, daß das P1-L9-Modul direkt mit der Thermostabilität von P RNAs in Zusammenhang steht – ein Befund, der einen wichtigen Schritt für das Verständnis der Beziehung von Struktur und Funktion katalytischer RNAs darstellt

Der Turm als Erfahrungsraum : die Ausstellung »AfE-Turm als Erfahrungsraum« und Publikation »Turmgeschichten« lässt den AfE-Turm wieder aufleben

Die Sprengung des AfE-Turms in Bockenheim stellte nicht nur eine stadtbildliche Veränderung dar. Für viele Menschen wurde damit auch ein Gebäude, das einen wichtigen Teil ihrer Biografie darstellt, gelöscht. Doch stellt die Sprengung nicht nur das Entfernen eines Symbols für einen bedeutungsvollen Lebensabschnitt vieler Menschen dar. Auch ein potenzieller Verlust vieler Geschichten geht damit einher