Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops

Evolution, Function and Structure of the Splicing Factor PRPF39

Beside 5’-capping and 3’-polyadenylation, splicing is one of the essential steps in the processing of most protein-coding genes in higher eukaryotes. It is catalyzed by the spliceosome, a large and dynamic RNA-protein molecular machine that encompasses five core components, the U1, U2, U4, U5 and U6 snRNPs. For each splicing reaction, a spliceosome is assembled anew in a stepwise manner. Spliceosomes must accurately recognize each splice site, as a single mistake can result in the production of a non-functional and potentially toxic protein. The crucial step of exon definition is facilitated by the U1 and U2 snRNP during early splicing. Cryo electron microscopy structures of early spliceosomal complexes in yeast have shown that the Prp39/Prp42 heterodimer is a crucial scaffolding subcomplex. It acts as a hub for multiple protein-protein interactions for example the contact between the U1 and the U2 snRNP, indicating that the Prp39/Prp42 heterodimer is important for the precise spatial positioning of the U1 and U2 snRNPs relative to each other. Interestingly there is no homolog for Prp42 in higher eukaryotes. PRPF39 is largely unstudied in higher eukaryotes and came to our attention because it is alternatively spliced in a differential manner in murine naïve vs. memory T-cells. I could show that an alternative exon is included in a differential manner. This can control PRPF39 expression by NMD in a tissue- and activation-dependent manner in mice and human, suggesting a role in adapting splicing efficiency to cell type specific requirements. Furthermore, I solved the crystal structure of murine PRPF39 at 3.3 Å resolution. The protein is largely α-helical and the structure shows the protein to be organized as a homodimer. Dimerization in solution could be confirmed with further biophysical assays. The mode of PRPF39 homodimerization is strikingly similar to heterodimerization of Prp39 and Prp42 in yeast. Structure guided point mutations could completely abolish dimerization and by using the monomeric PRPF39 mutants I could show that the monomer has a detrimental effect on splicing in vitro. Based on a structural comparison of murine PRPF39 and the yeast heterodimer, we performed a phylogenetic analysis showing, that organisms with a Prp39 homodimer have a substantially shortened U1 snRNA compared to organisms with a Prp39/Prp42 heterodimer. Our analysis indicates that a shortened U1 snRNA accompanied by a PRPF39 homodimer was crucial in the evolutionary development of more complex splicing. This observation is unexpected, as fewer splicing factors usually mirror lower splicing complexity and not the opposite. Taken together, my results reveal the structural and functional implications of murine PRPF39 on splicing. The data suggests, that a PRPF39 homodimer acts to substitute the Prp39/Prp42 heterodimer observed in yeast. Additionally, the reduction in RNA and protein complexity of the U1 snRNP may have been crucial in allowing highly complex and sophisticated splicing regulation across species

Molecular motions at the 5 stem-loop of U4 snRNA: Implications for U4/U6 snRNP assembly

Das humane 15.5K Protein bindet an die 5' Schleife der U4 snRNA (KtU4). Es begünstigt den Aufbau des Spliceosoms U4/U6 snRNP und ist notwendig für die Zusammenführung des 61K Proteins und des 20/60/90K Proteinkomplexes mit der U4-snRNA. In der kristallografischen Struktur des 15.5K-U4 snRNA Komplexes gehört die RNA Faltung zu der Familie der so genannten kink-turn (K-turn) Motive. Dieses Motiv ist durch einen scharfen Knick im Phosphodiesterrückgrat gekennzeichnet. Zwei Helixstämme sind durch eine purinreiche, asymmetrische, innere Schleife verbunden. Diese enthält eine herausgeklappte Uridinbase und zwei aufeinander folgende verschobene G-A Basenpaare. Der kürzere Helixstamm ist mit einer externen fünffachen Schleife verbunden. Mit Hilfe von Molekulardynamiksimulationen konnte ich zeigen, dass die Faltung von KtU4 durch die Proteinbindung unterstützt wird. Konformationsänderungen wie die Umwandlung zwischen alternativen Purin-Stapelschemata, der Verlust von G-A Basenpaaren und die Öffnung des K-turns (k-e Bewegung) trat nur in den Simulationen der ungebundenen RNA auf. Diese Simulationen zeigen zum ersten Mal die Dynamik des K-turn Motivs mit atomarer Auflösung und finden sich in hervorragender Übereinstimmung mit experimentellen Resultaten, die durch chemische Erprobung und FRET-Studien an Einzelmolekülen der RNA gesammelt wurden. In der ungebundenen RNA wurde die k-e Bewegung gleichermaßen durch den Verlust der G-A Basenpaare in der inneren Schleife und durch Flexibilität im Phosphatrückgrat der beiden Helixstämme hervorgerufen. Jedoch war der Verlust der G-A Basenpaare allein nicht ausreichend, um eine große Entfaltung der ungebundenen RNA zu erreichen. Eine Untersuchung der Eigenmodi der RNA Bewegung konnte zeigen, dass der Verlust der G-A Basenpaare nur mit dem ersten Eigenmodus korreliert ist aber nicht mit dem dritten Eigenmodus, der die k-e Bewegung beschreibt. Aufgrund dieser Ergebnisse komme ich zu dem Schluss, dass sich die G-A Basenpaare erst bei der Bindung an das 15.5K Protein bilden und d adurch selektiv die Ausrichtung der Helixstämme stabilisieren. Die externe Schleife konnte in der Kristallstruktur des 15.5K-KtU4 Komplexes nicht aufgelöst werden. In den Simulationen des Komplexes nahm sie eine bestimmte Orientierung ein, die nicht in der ungebundenen RNA beobachtet wurde. Sie trat auch dann nicht auf, wenn die natürliche Sequenz durch andere externe Schleifen ersetzt oder die Helixstämme verlängert wurden. Auf Grund der Simulationsergebnisse schlage ich vor, dass die nicht vorhandene Stapelung zwischen dem letzten Basenpaar des Helixstamms und dem benachbarten Nukleotid in der externen Schleife wichtig ist für die korrekte Faltung der RNA und eine wichtige Rolle in der nachfolgenden Bindung des 61K Proteins an die U4 snRNA spielt

Pre-mRNA Splicing: An Evolutionary Computational Journey from Ribozymes to Spliceosome

The intron\u2013exon organization of the genes is nowadays taken for granted and constitutes a fully established theory. DNA protein-coding sequences (exons) are not contiguous but rather separated by silent intervening fragments (introns), which must be removed in a process called pre-mRNA splicing. However, this fragmented composition of the eukaryotic genome has ancient origins. It appears that, during the initial stages of eukaryotic evolution, group II introns, i.e. self-splicing catalytic ribozymes, invaded the eukaryotic genome via the endosymbiosis of an alpha-proteobacterium in an archaeal host. At a later time, they split into the inert spliceosomal introns and the catalytically active small nuclear (sn)RNAs, which, together with additional splicing factors, gave rise to the eukaryotic spliceosome. This marked the transition from the autocatalytic splicing, mediated by ribozymes (RNA filaments endowed with an intrinsic catalytic activity) to splicing mediated by a protein-RNA machinery, the spliceosome. In the present thesis, the evolutionary relationship between group II introns and the spliceosome is retraced from a computational perspective by means of classical molecular dynamics simulations (MD), quantum mechanics calculations (QM) and combined quantum-classical simulations (QM/MM). The splicing process of these two different \u2013 but mechanistically related \u2013 large and sophisticated biomolecules is investigated with the aim of deciphering the reactivity and the structural properties from a computational point of view, with a focus on the role played by the Mg2+ ions as splicing cofactors. In Chapter 2, the importance of Mg2+ ions in the RNA biology is introduced. Not only they participate to the catalysis, but also represent essential structural and functional elements for RNA filaments. Moreover, the structural and molecular biology of group II intron ribozymes and the spliceosome machinery are widely discussed with a focus on their evolutionary links. Chapter 3 consists of a brief review of all the computational techniques employed in this thesis, from classical MD to QM and QM/MM simulations and enhanced sampling methods aimed at reconstructing the free energy of a process. Chapter 4 is entirely dedicated to the splicing mechanism promoted by group II intron ribozymes, representing the starting point of the evolutionary journey. In this chapter, a QM/MM study of the molecular mechanism of group II introns first-step hydrolytic splicing is presented, unveiling an RNA-adapted Steitz and Steitz\u2019s two-Mg2+-ion dissociative catalysis which differs from the one observed in protein enzymes. Chapter 5 is focused on Mg2+ ions, which are the natural cofactors of splicing, both in group II introns and in the spliceosome. Mg2+/RNA interplay is here addressed using a group II intron as a prototype of a large RNA molecule binding Mg2+. The performances of five different force fields currently used to describe Mg2+ in MD simulations are benchmarked, showing strengths and drawbacks. Moreover, the non-trivial electronic effects induced by Mg2+ on its ligands, such as charge transfer and polarization, are also characterized using 16 recurrent binding motifs. Overall, the study offers some guidelines on Mg2+ force fields for users and developers. Chapter 6 represents the final stop of the evolutionary journey. Here, an exquisite cryo-EM model of the ILS spliceosomal complex solved at 3.6 \uc5 resolution is used for a long-time scale MD study. This provides precious insights on the main proteins and snRNAs involved in the pre-mRNA splicing in eukaryotes as well as on the catalytic site. Unprecedentedly, the structural and dynamical properties of the spliceosome machinery are investigated at the atomistic level, with a particular emphasis on protein/RNA interplay through the characterization of their principal motions, among which the intron lariat/U2 snRNA helix unwinding

Identification of the Sm N protein and studies on its expression

The major species of mammalian Sm proteins associate with snRNA molecules to form small nuclear ribonucleoprotein particles (snRNPs) which are essential for pre-mRNA splicing. A series of immunoblots were probed with an anti-Sm monoclonal antibody. These experiments led to the identification of a cell-specific 28kDa protein called Sm N. The expression of the Sm N protein is restricted to cell lines and tissues which have the ability to utilise the alternative splicing pathway of the calcitonin/CGRP gene. This correlation suggests that the Sm N protein may play a role in determining the use of this alternative splicing pathway. A Sm N cDNA clone was isolated by immunoscreening a HeLa λgt11 expression library. Characterisation of the cDNA clone showed that the Sm N protein consists of 240 amino acids. It has a proline-rich carboxyl terminus and it is closely related to the Sm B and B' proteins. Northern blot analysis revealed that the Sm N protein is encoded by a 1.6kb mRNA transcript. RNA analysis showed that the Sm N gene is differentially expressed in HeLa cell lines. The levels of the Sm N protein and mRNA were shown to decline during the differentiation of embryonal carcinoma stem cells to parietal endoderm-like cells. Furthermore, the levels of the Sm N protein decline during the differentiation of embryonal stem cells suggesting that this effect may occur in vivo. The sera of some patients with the autoimmune disease systemic lupus erythematosus (SLE) contain anti-Sm autoantibodies. The anti-Sm monoclonal antibody which was used to identify Sm N recognises a disease autoepitope. In order to localise this epitope on the Sm N protein, a short, 135 nucleotide Sm N clone was obtained by immunoscreening a λgt11 expression library. A lysogen of this clone was used to detect the presence of anti-Sm antibodies in SLE sera. It was found that both anti-Sm and anti-RNP autoantibodies reacted with the fusion protein. This effect was due to amino acid sequence similarities between Sm N and the U1 snRNP-specific C protein. Immunoblotting analysis was used to show that the highly immunoreactive Sm B, B', and D proteins increase in abundance in Vero cells infected with herpes simplex virus type 2. This effect may increase the antigenicity of these Sm proteins

ModeRNA: a tool for comparative modeling of RNA 3D structure

RNA is a large group of functionally important biomacromolecules. In striking analogy to proteins, the function of RNA depends on its structure and dynamics, which in turn is encoded in the linear sequence. However, while there are numerous methods for computational prediction of protein three-dimensional (3D) structure from sequence, with comparative modeling being the most reliable approach, there are very few such methods for RNA. Here, we present ModeRNA, a software tool for comparative modeling of RNA 3D structures. As an input, ModeRNA requires a 3D structure of a template RNA molecule, and a sequence alignment between the target to be modeled and the template. It must be emphasized that a good alignment is required for successful modeling, and for large and complex RNA molecules the development of a good alignment usually requires manual adjustments of the input data based on previous expertise of the respective RNA family. ModeRNA can model post-transcriptional modifications, a functionally important feature analogous to post-translational modifications in proteins. ModeRNA can also model DNA structures or use them as templates. It is equipped with many functions for merging fragments of different nucleic acid structures into a single model and analyzing their geometry. Windows and UNIX implementations of ModeRNA with comprehensive documentation and a tutorial are freely available

Structure-Function Relationships of Long Non-Coding RNAs in Living Cells

From the beginning of the era of molecular biology in the 1960s until the 1980s, RNA was widely regarded as a passive cellular messenger. However, the importance of RNA has been steadily emerging over the last 30 years and we now know that it is often a critical and central component of genetic regulation. Recently, long non-coding RNAs (lncRNA) have become the focus of intense research because of their roles in development and disease. For most functional RNAs, complex structural characteristics underlie the biological function of the molecule. However, the difficulty of de novo RNA structure prediction and the relatively low abundance of lncRNA transcripts have been roadblocks to experimental structure probing. As a result, very little is known about the structural features of lncRNAs. In this work, I present experimental and analytical methods that enable chemical structure probing of rare RNA transcripts and identification of stable RNA-protein interaction sites. First, I show that polymerase chain reactions can be used as an enrichment strategy that faithfully maintains structure-probing data. I then outline an analytical framework that enables statistically rigorous detection of RNA-protein interactions in living cells. Finally, I apply these new methodologies to the Xist lncRNA and present a data-driven secondary structure model that highlights the extensive structures present throughout the transcript. I then identify nearly 200 specific sites where Xist is strongly impacted by the cellular environment and use them to identify several new protein interaction domains within Xist. Together, this work provides new experimental and analytical tools, as well as many new insights on the relationship between lncRNA structure and function, that will enable rapid study of lncRNA structures in the future.Doctor of Philosoph

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

Characterizing Stargardt disease-causing mutations to identify ABCA4 gene lesions amenable to splice intervention therapeutics

Stargardt disease (STGD1, OMIM: 248200) is an autosomal recessive retinal dystrophy, characterized by bilateral progressive central vision loss and subretinal deposition of lipofuscin-like substances. The wide spectrum of clinical phenotypes, ranging from childhood-onset cone-rod dystrophy to late-onset macular pattern dystrophy-like disease, indicates a more complex genotype-phenotype correlation than previously believed. The association of mutations in the ATP-binding cassette transporter gene, ABCA4, with STGD1 was first reported in two families in 1997. The ABCA4 protein encoded by ABCA4 is predominantly expressed in outer segments of photoreceptors and retinal pigment epithelial (RPE) cells in retina..