Structural and functional analysis of eukaryotic snoRNP complexes catalyzing 2’-O-ribose methylation of rRNA

Translating the information encoded in messenger RNAs (mRNAs) into functional proteins is an essential cellular process carried out by large molecular machines termed ribosomes. Ribosomes are large ribonucleoprotein (RNP) particles, whose biogenesis is an energetically demanding and highly regulated process. During the early stages of ribosome biogenesis, the ribosomal RNAs (rRNAs) get covalently modified. One of the most abundant of these covalent modifications is the methylation of the 2’ hydroxyl group of the ribose (2’-O-Me) in specific nucleotides of the rRNA. Many of these 2’-O-methylated sites are located in functionally important regions of the matured ribosome, such as the peptidyl transferase center (PTC) or the decoding center. Unsurprisingly, aberrations in 2’-O-Me are associated with pathological developments such as cancer and neurological diseases in human. In archaea and eukaryotes 2’-O-Me modifications on rRNA are transferred by the Box C/D enzymes, which are multi-component RNPs that use guide RNAs to mediate site specific 2’-O-methylation on rRNA. Most of the available structural and functional data on the Box C/D RNP enzymes are based on the archaeal enzyme. Conversely, only little is known on the structural and functional details of the eukaryotic Box C/D enzyme. Therefore, the archaeal system is being used as a structural and functional proxy for the eukaryotic enzyme. To expand to structural and functional knowledge about the eukaryotic Box C/D small nucleolar ribonucleoprotein (snoRNP) enzymes and examine the validity of the archaeal enzymes as a proxy I used a combination of biochemical, analytical and structural methods to analyze and characterize two subcomplexes of the eukaryotic Box C/D snoRNP from S. cerevisiae in vitro. Using fluorescence-based electrophoretic mobility shift assays I could characterize the binding requirements and affinities between the eukaryotic and archaeal Box C/D primary RNA-binding protein Snu13 and L7Ae, respectively, and the lesser conserved of two protein binding motifs on the Box C/D guide RNA. I also present the first high-resolution structure of archaeal L7Ae in complex with a non-standard Box C/D protein binding motif solved by X-ray crystallography. Using a combination of quantitative mass spectrometry, multi-angle light scattering and radioactivity-based enzymatic assays I determined the stoichiometries of in vitro reconstituted chimeric Box C/D enzymes based on different guide RNAs, demonstrating a potentially different structural arrangement in eukaryotic Box C/D enzymes as compared to archaeal enzymes. Lastly, the high-resolution structure of the eukaryotic 2’-O-methyltransferase Nop1 in complex with the scaffolding protein Nop56 from the Box C/D enzyme solved by X-ray crystallography highlights significant differences to the archaeal orthologs. The presented data expands the structural and functional information on the eukaryotic Box C/D and suggest together with exiting literature substantial differences between eukaryotic and archaeal Box C/D enzymes.Die Übersetzung der in Messenger-RNAs kodierten Informationen in Proteine ist ein wesentlicher zellulärer Prozess, der von Ribosomen ausgeführt wird. Ribosomen sind große Ribonukleoprotein (RNP) -Partikel, deren Biogenese ein energetisch anspruchsvoller und stark regulierter Prozess ist. In den frühen Stadien der Ribosomenbiogenese werden die ribosomalen RNAs kovalent modifiziert. Eine der am häufigsten vorkommenden dieser kovalenten Modifikationen ist die Methylierung der 2'-Hydroxylgruppe der Ribose (2'-O-Me) in spezifischen Nukleotiden der rRNA. Viele dieser 2'-O-methylierten Stellen befinden sich in funktionell wichtigen Regionen des Ribosoms. Aberrationen bei 2'-O-Me sind mit Krebs und neurologischen Erkrankungen beim Menschen verbunden. In Archaeen und Eukaryoten werden 2'-O-Me-Modifikationen auf rRNA durch die Box C/D-Enzyme übertragen, bei denen es sich um Mehrkomponenten-RNPs handelt, die Leit-RNAs verwenden, um positionsspezifische 2'-O-Methylierung auf rRNA zu vermitteln. Der Großteil der verfügbaren Daten zu den Box C/D RNP-Enzymen basiert auf dem archaealen Enzymen und nur wenig ist über die strukturellen und funktionellen Details des eukaryotischen Box C/D-Enzyms bekannt, weswegen das archaeale System als Modell für das eukaryotische Enzym verwendet wird. Um das Wissen über die eukaryotischen Box C/D Enzyme zu erweitern und die Gültigkeit der archaealen Enzyme als Modell zu untersuchen, verwendete ich eine Kombination aus biochemischen, analytischen und strukturellen Methoden, um zwei Subkomplexe des eukaryotische Box C/D Enzyms von S. cerevisiae in vitro zu charakterisieren. Unter Verwendung fluoreszenzbasierter elektrophoretischer Mobilitätsverschiebungstests konnte ich die Bindungsanforderungen und -affinitäten zwischen dem eukaryotischen und dem archaealen Box C/D-RNA-Bindeprotein Snu13 bzw. L7Ae und dem weniger konservierten von zwei Proteinbindungsmotiven auf der Box C/D Leit-RNA charakterisieren. Ich präsentiere die erste hochauflösende Struktur von archaealem L7Ae im Komplex mit einem nicht standardmäßigen Box C/D Proteinbindungsmotiv, gelöst mit Röntgenkristallographie. Unter Verwendung einer Kombination aus quantitativer Massenspektrometrie, Mehrwinkellichtstreuung und enzymatischen Assays bestimmte ich die Proteinstöchiometrien von in vitro rekonstituierten chimären Box C/D-Enzymen und zeige eine möglicherweise unterschiedliche strukturelle Anordnung in eukaryotischen Box C/D-Enzymen im Vergleich zu archaealen Enzymen. Die hochauflösende Struktur der 2'-O-Methyltransferase Nop1 im Komplex mit Nop56 aus dem eukaryotischen Box C/D-Enzym zeigt signifikante Unterschiede zu den archaealen Orthologen. Die präsentierten Daten erweitern das strukturelle und funktionelle Wissen über eukaryotische Box C/D Enzyme und legen zusammen mit der vorhandenen Literatur erhebliche Unterschiede zwischen eukaryotischen und archaealen Box C/D-Enzymen da

Eukaryotic Box C/D methylation machinery has two non-symmetric protein assembly sites

Box C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2’-OH of RNA. The central ‘guide RNA’ has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C’/D’ motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C’/D’ motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2’-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C’/D’ motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2’-O-methylation enzymes

Insights into the modulation of the interaction between the Unique and SH3 domains in Src Family kinases

Die Fyn-related Kinase (FRK) oder Rak ist eines der besser erforschten Mitglieder einer Familie von Nicht-Rezeptor Tyrosinkinasen, die sich aus FRK, PTK6/Brk (Breast Tumor Kinase) und SRMS (Src-related tyrosine kinase lacking C-terminal regulatory and N-terminal myristoylation sites) zusammensetzen. Im Gegensatz zu den strukturell nahe verwandten Src Family Kinasen (SFKs) haben die Mitglieder der FRK/PTK6 Familie keine N-terminale Lipidierung (Myristoylierung und/oder Palmitoylierung) und ebenso fehlt ihnen die Src-homolgy 4 (SH4) Domäne. Beiden Kinasefamilien sind die intrinsically disordered Unique Domäne, die Src-homolgy 3 und 2 (SH3 and SH2) Domänen sowie die C-terminale Kinasedomäne gemein. Vorangegangene Untersuchungen der intrinsically disordered Unique Domäne von c-Src zeigten, dass diese Domäne mit der anschlie\textbeta enden SH3 Domäne und der cytoplasmatischen Seite der Zellmembran unter der Formation einer Peptidschlaufe innerhalb der Domäne interagiert. Diese Schlaufenbildung wurde u.a. auch in Fyn, einer weitern Src Family Kinase, beobachtet. FRK besitzt zwei Cysteine in der Primärstruktur seiner Unique Domäne die in Regionen sind in denen sich konservierte aromatische Aminosäuren in den SFKs befinden, welche in die Formation der Peptidschlaufe involviert sind. Daher liegt die Hypothese nahe, dass diese beiden Cysteine an einer ähnlichen Schlaufenformation in FRK beteiligt sind, die zum Beispiel durch die Chelatierung von Metalionen zwischen den beiden Cysteinen bewirkt werden könnte. Im Rahmen dieser Arbeit werden die erste Expression sowie Aufreinigung des N-terminalen Teils des humanen FRK Proteins, bestehend aus Unique und SH3 Domäne, beschrieben. Dieses Konstrukt wird in dieser Arbeit als FRK(USH3) bezeichnet.Ein vollständiges Protokoll, bestehend aus der Expression in E.coli, erfolgreichem Falten eines unlöslich-exprimierten Proteinkonstrukts und der Aufreinigung eines zur Aggregation neigendem Proteins für die Analyse über NMR Spektroskopie,wurde entwickelt. Des Weiteren wird das erste 1H-15N zwei-dimensionale NMR Spektrum von FRK(USH3) präsentiert. Es konnte gezeigt werden, dass der N-terminal Teil von FRK bei Proteinkonzentrationen höher als 100M eine starke Tendenz zur Aggregation zeigt und daher nur in einem sehr niedrigen Konzentrationsbereich für die Analyse mittels NMR Spektroskopie verwendet werden kann. Derivatisierungsversuche, die in einer Maskierung der Thiolgruppen in den Cysteinseitenketten und der Erhöhung der Nettoladung des Konstrukts resultierten, konnten die Löslichkeit geringfügig erhöhen und die Aggregationstendenz senken. Dadurch konnte die Qualität der aufgenommenen NMR Spektren verbessert werden. Dynamic Light Scattering (DLS) und Native Polyacrylamidgelelektrophorese wurden verwendet um Unterschiede zwischen dem derivatisierten und nicht-derivatisierten Konstrukt zu analysieren.The Fyn-related Kinase (FRK) or Rak is one of the leading members of a distinct family of non-receptor tyrosine kinases consisting of FRK, PTK6 or Brk (Breast Tumer Kinase) and SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites). In contrast to the structurally closely related Src Family Kinases (SFKs), members of the FRK/PTK6 family lack the N-terminal lipidation (myristoylation and/or palmitoylation) and also the Src-homology 4 (SH4) domain. Both families share the intrinsically disordered Unique domain, the Src-homology 3 and 2 (SH3 and SH2) domains and the C-terminal kinase domain. Previous research on the intrinsically disordered Unique domain of c-Src revealed that the Unique domain interacts with the SH3 domain and the cytoplasmatic lipid layer of the cellular membrane under the formation of a peptide loop within the Unique domain. This loop formation has also been observed in Fyn, another SFK. FRK naturally provides two cysteine residues in regions of the Unique domain that are involved in the loop formation in c-Src and which could therefere be involved in a loop formation by metal ion chelation or other mechanisms. In this work the first expression and purification of the N-terminal part of human FRK, containing Unique and SH3 domain, referred to as FRK(USH3), is described. A complete protocol involving expression in E. coli, successful refolding of an insoluble expressed construct and the purification of an aggregation prone protein for analysis by NMR spectroscopy was developed. Furthermore the first 1H-15N two-dimensional NMR spectrum of FRK(USH3) is reported

High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain

Ribosomal RNA (rRNA) carries extensive 2′-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2′-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein–protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes

High-resolution structure of eukaryotic Fibrillarin interacting with Nop56 amino-terminal domain.

Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes

Evaluation of colon cancer histomorphology: a comparison between formalin and PAXgene tissue fixation by an international ring trial.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41-0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67-1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis