Roles of the novel 5'-Polynucleotide kinase NoI9 in ribosome synthesis

In einer sich teilenden Zelle nehmen ribosomale RNAs 80% der gesamten zellulären RNA ein. Sie sind die Hauptkomponente der Ribosomen, grossen Komplexen aus RNA und Proteinen, die als “Protein-Fabriken” der Zelle eine zentrale Aufgabe ausüben. Die korrekte und effiziente Biosynthese der ribosomalen RNA ist daher grundlegend für jede Zelle. Im Nukleolus werden drei der vier ribosomalen RNAs (rRNAs), 18S, 5.8S und 28S, gemeinsam von der RNA Polymerase I zu einem einzelnen polyzistronen Precursor transkribiert. In einer geordneten Aufeinanderfolge von endo- und exonukleo-lytischen Aktivitäten maturieren diese rRNAs und werden letztendlich gemeinsam mit der 5S rRNA und einer Vielzahl von ribosomalen Proteinen zum Ribosom zusammengefügt. Während meiner Doktorarbeit habe ich die erste nukleoläre Polynukleotid-Kinase Nol9/Grc3 identifiziert und ihre Funktion studiert. Nol9/Grc3 ist in zweierlei Hinsicht an der Maturierung von ribosomalen RNAs beteiligt: 1. S. cerevisiae Grc3 ist essenziell für eine effiziente Beendigung der Transkription von rRNA Genen als Teil des Rat1-abhängigen ‘Torpedo-Mechanismus” und somit wesentlich für die rasche Wiederverwendung der RNA Polymerase I. 2. Humanes Nol9 hat eine Funktion bei der Prozessierung der 5.8S und 28S ribosomalen RNA aus ihrem gemeinsamen Precursor, möglicherweise in einem Xrn2-abhängigen Prozessierungsschritt. Diese Ergebnisse beleuchten ein moegliches Zusammenspiel der Polynukleotid-Kinase Nol9/Grc3 und der 5’-3’ Exonuklease Xrn2/Rat1. Berücksichtigt man die wachsenden Berichte über zusätzliche Aufgaben des Nukleolus im RNA Metabolismus abseits der Maturierung ribosomaler RNA und die Aktivitaet von Nol9/Grc3 an sowohl einzel- als auch doppelsträngigen RNA- und DNA-Substraten, zeichnen sich mögliche weitere Funktionen für die Polynukleotid-Kinase Nol9/Grc3 ab.In a dividing cell, about 80% of the cellular RNA consist of ribosomal RNAs (rRNAs), the core components of the ribosomes. Efficient protein production relies on the sufficient availability of ribosomes; therefore indefectible rRNA processing is fundamental to every cell. The 18S, 5.8S and 28S rRNAs are transcribed in the nucleolus by RNA polymerase I as a single polycistronic precursor RNA and liberated by a complex series of endo- and exonucleolytic cleavage events. Eventually, they are assembled together with the 5S rRNA and a plethora of ribosomal proteins to form the ribosomes. In my PhD work, I identified the first nucleolar polynucleotide kinase Nol9/Grc3 and investigated its involvement in two different aspects of rRNA biogenesis: 1. RNA Polymerase I transcription termination; and 2. processing of the large subunit rRNAs. In yeast, Grc3 is essential for efficient transcription termination in the Rat1-dependent ‘torpedo mechanism’ thereby enabling rapid recycling of RNA polymerase I. In human cells, Nol9 is required for the processing of 5.8S and 28S rRNAs, the components of the large 60S ribosomal subunits, most likely at a Xrn2-dependent processing step. These findings intrigue a potential interplay between the polynucleotide kinase Nol9/Grc3 and the 5’-3’ exonuclease Xrn2/Rat1. Given the involvement of the nucleolus in a growing number of RNA metabolic pathways and the fact that Nol9/Grc3 can efficiently phosphorylate single and double stranded RNA and DNA substrates, Nol9/Grc3 potentially has additional roles in RNA metabolism

Nol9 is a novel polynucleotide 5′-kinase involved in ribosomal RNA processing

The production and processing of ribosomal RNA is an essential and complex process. Here, a polynucleotide 5′-kinase, Nol9, is shown to have an important function in pre-rRNA processing and 60S ribosomal subunit biogenesis

Nanoenviroments of the β-Subunit of L-Type Voltage-Gated Calcium Channels in Adult Cardiomyocytes

In cardiomyocytes, Ca2+ influx through L-type voltage-gated calcium channels (LTCCs) following membrane depolarization regulates crucial Ca2+-dependent processes including duration and amplitude of the action potentials and excitation-contraction coupling. LTCCs are heteromultimeric proteins composed of the Cavα1, Cavβ, Cavα2δ and Cavγ subunits. Here, using ascorbate peroxidase (APEX2)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nanoenvironments of Cavβ2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, cardiac contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays and co-immunoprecipitation analyses revealed that Cavβ2 interacts with the ryanodine receptor 2 (RyR2) in adult cardiomyocytes, probably coupling LTCCs and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src-homology 3 domain of Cavβ2 and is necessary for an effective pacing frequency-dependent increase of the Ca2+-induced Ca2+ release mechanism in cardiomyocytes

The β2_{2}-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy

L-type voltage-gated calcium channels (LTCCs) regulate crucial physiological processes in the heart. They are composed of the Cavα1 pore-forming subunit and the accessory subunits Cavβ, Cavα2δ, and Cavγ. Cavβ is a cytosolic protein that regulates channel trafficking and activity, but it also exerts other LTCC-independent functions. Cardiac hypertrophy, a relevant risk factor for the development of congestive heart failure, depends on the activation of calcium-dependent pro-hypertrophic signaling cascades. Here, by using shRNA-mediated Cavβ silencing, we demonstrate that Cavβ2 downregulation enhances α1-adrenergic receptor agonist-induced cardiomyocyte hypertrophy. We report that a pool of Cavβ2 is targeted to the nucleus in cardiomyocytes and that the expression of this nuclear fraction decreases during in vitro and in vivo induction of cardiac hypertrophy. Moreover, the overexpression of nucleus-targeted Cavβ2 in cardiomyocytes inhibits in vitro-induced hypertrophy. Quantitative proteomic analyses showed that Cavβ2 knockdown leads to changes in the expression of diverse myocyte proteins, including reduction of calpastatin, an endogenous inhibitor of the calcium-dependent protease calpain. Accordingly, Cavβ2-downregulated cardiomyocytes had a 2-fold increase in calpain activity as compared to control cells. Furthermore, inhibition of calpain activity in Cavβ2-downregulated cells abolished the enhanced α1-adrenergic receptor agonist-induced hypertrophy observed in these cells. Our findings indicate that in cardiomyocytes, a nuclear pool of Cavβ2 participates in cellular functions that are independent of LTCC activity. They also indicate that a downregulation of nuclear Cavβ2 during cardiomyocyte hypertrophy promotes the activation of calpain-dependent hypertrophic pathways