Alors que le complexe snRNP est bien décrit chez les vertébrés, il nécessite plus d’études chez les invertébrés. Le snARN 7SK sert de maintient structural pour la fixation d’HEXIM à P-TEFb. En retour, HEXIM inhibe l’activité kinase de CDK9 via une fixation directe avec la Cycline T. En conséquence, les interactions entre le snARN 7SK et HEXIM va piéger le complexe P-TEFb sous une forme inactive qui conduit à inhiber l’élongation transcriptionnelle. Dans notre étude, nous montrons qu’un contrôle de l’activité P-TEFb existe aussi chez la Drosophile. Et la dynamique d’équilibre entre les deux formes de P-TEFb dépend également du snARN 7SK. Ce modèle est donc utilisé pour étudier le rôle biologique de la snRNP, et plus spécialement d’HEXIM, dans un contexte intégré. Nous avons donc analysé le profile d’expression d’HEXM durant le cycle de vie de la Drosophile et plus particulièrement pendant l’embryogenèse et l’organogenèse. L’expression permanente et ubiquitaire d’HEXIM suggère qu’elle est nécessaire au développement. Le fait que la perte de fonction d’HEXIM mène à de nombreux et sévères défauts confirme cette hypothèse. En utilisant le modèle des disques imaginaux de l’aile et de l’œil, nous avons étudié plus en profondeur le rôle d’HEXIM et nous avons montré qu’elle est essentielle pour la viabilité cellulaire. De plus, la perte de fonction d’HEXIM conduit à des changements du destin cellulaire et à des modifications des profiles d’expression de plusieurs gènes sélecteurs ou de morphogènes. De façon surprenante, la diminution d’HEXIM induit l’accumulation de Ci155 qui est requise pour activer l’expression de Ptc, ainsi que l’activation ectopique de la voie Hh. Cette accumulation notable de Ci155 est également détectée dans les cellules “immortelles” et dans les tissus en cours de régénération à la suite d’une ablation par voie génétique. Sur la base de ces données, nous proposons un rôle possible de l’accumulation de Ci155 dans le phénomène de prolifération compensatrice. Finalement, nous avons caractérisé un nouvel analogue du snARN 7SK chez la Drosophile, qui a été nommé dm7SK-like snARN. Ce dernier a une structure secondaire très similaire à celle de ces homologues vertébrés, alors que la séquence primaire est assez différente. De plus, presque tous les domaines structuraux importants pour les interactions avec HEXIM et les autres partenaires sont conservés chez cet ARN. Des interactions directes ont été démontrées entre HEXIM et cet ARN suggérant qu’il est un analogue structural du snARN 7SK. Ainsi, la présence de deux analogues du snARN 7SK suggère un autre niveau de régulation de l’expression des gènes, au moins chez la Drosophile. / Whereas 7SK snRNP complex has been well characterized in vertebrates, its activities still remain to be further elucidated in invertebrates. 7SK snRNA serves as a structural scaffold for the efficient binding of HEXIM to P-TEFb. HEXIM in turn inhibits the kinase activity of CDK9 via its direct binding to CyclinT. Consequently, the interaction between 7SK snRNA and HEXIM sequesters the active P-TEFb complex into the inactive form, thereby suppressing the transcription elongation. In this study, we first show that a similar P-TEFb control system exists in Drosophila. In addition, the dynamic equilibrium of the two complexes of P-TEFb in Drosophila also depends on 7SK snRNA. Thank to this similarity, we are able to examine the biological role of 7SK snRNP complex, especially HEXIM protein, in an integrative organism as Drosophila model. We next document the expression profile of HEXIM throughout the life cycle of Drosophila, especially during embryogenesis and organogenesis. The continuous and ubiquitous expression of HEXIM suggests its necessity during development. We demonstrate that HEXIM is indeed essential for the proper development of Drosophila, since its down-regulation results in numerous severe defects. By using wing and eye imaginal discs as study models, we further examine biological roles of HEXIM, and reveal that it is required for cell viability. Moreover, HEXIM knockdown leads to changes in cell fate commitments, and modifications in expression patterns of several selector genes and morphogens. Strikingly, down-regulation of HEXIM significantly induces the accumulation of Ci155, which is required for Ptc expression, and the ectopic activation of Hh signaling. This remarkable accumulation of Ci155 is also detected in “undead cells” and regenerated tissue upon genetic ablation. Given these findings, we thus propose a putative role of Ci155 accumulation in compensatory proliferation. Finally, we characterize a novel analog of 7SK snRNA in Drosophila, which is named dm7SK-like snRNA. This snRNA displays a very similar secondary structure with its vertebrate homologs, although the primary sequence is relatively different. More importantly, almost all of the structural elements crucial for the interaction with HEXIM and other partners are found conserved in this novel dm7SK-like snRNA. A direct interaction between dHEXIM and this snRNA also suggests that it is a functional analog of 7SK snRNA in Drosophila. Thus, the intriguing finding of the two analogs of 7SK snRNA would propose another regulation level of gene expression, at least in Drosophila.

Regulation of transcription elongation by P-TEFb is critical for proper gene expression and cell survival. The cell possesses large quantities of P-TEFb, but the vast majority of it is locked away and inactive in the 7SK snRNP. Since the discovery of the 7SK snRNP, research has been conducted to determine how P-TEFb is released from this complex. The goal of the research presented in this thesis is to better understand how the 7SK snRNP regulates P-TEFb and ultimately, gene expression. This work documents the discovery and characterization of the 7SK stability protein LARP7. LARP7 is is associated with 7SK regardless of the presence of P-TEFb and HEXIM1. Stabilization of 7SK is essential for maintenance of the RNP because loss of LARP7 results in an increase in free P-TEFb and a significant reduction in the amount of 7SK. These results indicate that stabilization of the 7SK snRNP by LARP7 is important for regulating P-TEFb homeostasis. Although P-TEFb was first characterized from Drosophila lysates, the conservation of the 7SK snRNP and the mechanisms regulating P-TEFb inhibition have not been described. Here, the Drosophila melanogaster homologues of LARP7 and 7SK are characterized. These studies show that the system of P-TEFb regulation is similar in flies and this makes Drosophila an attractive model system for studying P-TEFb regulation through embryonic and larval development. Finally, factors and modifications involved in releasing P-TEFb directly are explored. An assay was developed for discovering proteins that can bind to and release P-TEFb from the 7SK snRNP. Use of this assay showed that post-translational modification of the components of the 7SK snRNP do not cause P-TEFb release directly. However, HIV Tat and the C-terminal P-TEFb binding region of the bromodomain containing protein, Brd4, are capable of extracting P-TEFb directly. Most importantly, the release of P-TEFb is followed by a conformational change in 7SK RNA that prevents the continued binding of HEXIM1 to the complex. P-TEFb release from the 7SK snRNP is the result of direct extraction of P-TEFb by viral or cellular proteins, and not post-translational modifications or a competition between HEXIM1 and hnRNP proteins for 7SK binding.

Genexpression in Eukaryoten beschreibt einen mehrstufigen Prozess, welcher auf Ebene der Transkription durch den positiven Transkriptionselongationsfaktor P-TEFb entscheidend reguliert wird. PTEFb bildet einen heterodimeren Komplex aus der Cyclin abhängigen Kinase 9 und deren Kofaktor Cyclin T1/2. Dieser Komplex aktiviert die Elongation der Transkription durch Phosphorylierung der negativen Elongationsfaktoren DSIF und NELF. Darüber hinaus phosphoryliert PTEFb Serin2 Reste in der C-terminalen Domäne von RNA PolII und stimuliert so die kotranskriptionelle Prozessierung der synthetisierten prä-mRNA. In Anpassung an unterschiedliche Wachstumsbedingungen wird die Aktivität dieses Faktors durch reversible Interaktion mit 7SK RNA und HEXIM Proteinen innerhalb eines katalytisch inaktiven Ribonukleoproteinpartikels (7SK RNP) streng kontrolliert. Dieses sensible Gleichgewicht zwischen P-TEFb auf der einen und dem 7SK RNP auf der anderen Seite bildet die Grundlage der Regulation der Transkriptionselongation. Trotz der hohen Abundanz von 7SK RNA in der Zelle, assoziiert in vivo jedoch nur ein relativ kleiner Teil hiervon mit P-TEFb, sodass die effektiv zur Verfügung stehende RNA-Menge für die Bildung des 7SK RNP vermutlich limitierend wirkt. Ziel der vorliegenden Arbeit war es daher neue 7SK RNA interagierende Faktoren zu identifizieren, welche die Interaktion von PTEFb mit dem 7SK RNP steuern und so die PolII abhängige Transkription regulieren. Anhand verschiedener chromatographischer Reinigungen konnte zunächst ein bislang uncharakterisiertes La ähnliches Protein (LARP7) mit einer spezifischen Affinität für Pyrimidinreiche RNAs isoliert werden. LARP7 bindet, wie durch immunbiochemische Analysen und RNA- Bindungsstudien gezeigt werden konnte, quantitativ an das hoch konservierte uridylreiche 3´- Ende von 7SK RNA. Diese Assoziation erfordert dessen La- und RRMDomänen und erhöht wesentlich die Stabilität der RNA. Darüber hinaus kofraktioniert LARP7 mit weiteren Faktoren des 7SK RNP, bindet direkt an HEXIM1 und P-TEFb und stellt somit ebenfalls eine integrale Komponente des 7SK RNP dar. Die gewonnenen Daten weisen außerdem erstmals darauf hin, dass P-TEFb durch einen vorgeformten trimeren Komplexes, bestehend aus HEXIM1, 7SK RNA und LARP7 inhibiert wird. Reportergenanalysen in TZMbl-Zellen, welche Luziferase unter der Kontrolle des streng P-TEFb abhängigen HIV-1-LTRPromotors exprimieren zeigten, dass diese Inhibition im Wesentlichen durch LARP7 vermittelt wird. So ließ sich nach Reduktion der LARP7 Expression mittels RNAi eine signifikante Steigerung der Transkription vom HIV-1-LTR-Promotor beobachten. Eine ähnliche Stimulation der Transkription von PolII konnte in LARP7 defizienten HeLa-Zellen durch quantitative Real-Time-PCR auch für eine Reihe zellulärer Gene nachgewiesen werden. Die Beobachtung, dass LARP7 die generelle PolII Transkription reprimiert, korreliert zudem mit einer bereits beschriebenen Tumorsupressorfunktion des LARP7 homologen mxc Proteins aus D. melanogaster. Somit beeinflusst LARP7 das zelluläre Gleichgewicht zwischen freiem und 7SK RNP-gebundenem P-TEFb und fungiert somit als negativer Regulator der PolII Transkription in vivo. / Eucaryotic gene expression is a multistep process, which is critically regulated on the level of RNA polII transcription by the positive transcription elongation factor P-TEFb. P-TEFb forms a heterodimeric complex, consisting of the cyclin-dependent kinase 9 and its cofactor cyclin T1/2. This complex stimulates transcriptional elongation as well as the cotranscriptional processing of the synthesized pre-mRNA by phosphorylation of negative elongation factors and the RNA polII Cterminal domain. To accommodate different growth conditions, P-TEFb activity is kept under tight control through its reversible interaction with 7SK RNA and HEXIM proteins in a catalytically inactive ribonucleoprotein particle (RNP). This sensitive balance between PTEFb on the one hand and the 7SK RNP on the other represents the basis of transcriptional regulation of elongation. Despite the high abundance of 7SK RNA in the cell, only a small part is associated with P-TEFb in vivo, suggesting that the actual amount of RNA available limits the formation of the 7SK RNP. Hence, the objective of the present study was to identify novel 7SK RNA interacting factors, which direct the interaction of P-TEFb with the 7SK RNP, thereby regulating polII dependent transcription. At first, using different chromatographic purification strategies, an as yet uncharacterized La related protein (LARP7) with an affinity to pyrimidine- rich RNAs was isolated. Immunobiochemical analysis and RNA binding studies revealed, that LARP7 quantitatively associates with the highly conserved 3´-UUU-OH terminus of 7SK RNA. This binding requires its La- and RRM-domain and dramatically increases RNA stability. Moreover, LARP7 co-fractionates with additional factors of the 7SK RNP, binds directly to HEXIM1 and P-TEFb and therefore likewise constitutes an integral component of the 7SK RNP. Data presented here indicate that P-TEFb is inhibited upon association with a trimeric complex consisting of HEXIM1, 7SK RNA and LARP7. Furthermore, reporter gene analysis in TZMbl cells - cells expressing luciferase under the control of the strictly P-TEFb dependent HIV-1-LTR promoter - demonstrated this inhibition to be mainly mediated by LARP7. Thus, reduction of LARP7 expression by RNA-interference led to a significant stimulation of transcription in TZMbl cells. In addition, quantitative real time pcr revealed a similar effect on transcription for a series of cellular genes in LARP7 deficient HeLa cells. Moreover, the observation, that LARP7 represses polII transcription in general correlates well with a known function of the d. melanogaster LARP7 homologue mxc as a tumor suppressor. Thus, LARP7 affects the cellular P-TEFb/7SK RNP equilibrium und serves as a negative regulator of polII transcription in vivo.

In mammals, a major fraction of the genome is transcribed as non-coding RNAs. An increasing amount of evidence has accumulated showing that non-coding RNAs play important roles both for normal cell function and in disease processes such as cancer or neurodegeneration. Interpreting the functions of non-coding RNAs and the molecular mechanisms through which they act is one of the most important challenges facing RNA biology today. In my Ph.D. thesis, I have been investigating the role of 7SK, one of the most abundant non-coding RNAs, in the development and function of motoneurons. 7SK is a highly structured 331 nt RNA transcribed by RNA polymerase III. It forms four stem-loop (SL) structures that serve as binding sites for different proteins. Larp7 binds to SL4 and protects the 3' end from exonucleolytic degradation. SL1 serves as a binding site for HEXIM1, which recruits the pTEFb complex composed of CDK9 and cyclin T1. pTEFb has a stimulatory role for transcription and is regulated through sequestration by 7SK. More recently, a number of heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified as 7SK interactors. One of these is hnRNP R, which has been shown to have a role in motoneuron development by regulating axon growth. Taken together, 7SK’s function involves interactions with RNA binding proteins, and different RNA binding proteins interact with different regions of 7SK, such that 7SK can be considered as a hub for recruitment and release of different proteins. The questions I have addressed during my Ph.D. are as follows: 1) which region of 7SK interacts with hnRNP R, a main interactor of 7SK? 2) What effects occur in motoneurons after the protein binding sites of 7SK are abolished? 3) Are there additional 7SK binding proteins that regulate the functions of the 7SK RNP? Using in vitro and in vivo experiments, I found that hnRNP R binds both the SL1 and SL3 region of 7SK, and also that pTEFb cannot be recruited after deleting the SL1 region but is able to bind to a 7SK mutant with deletion of SL3. In order to answer the question of how the 7SK mutations affect axon outgrowth and elongation in mouse primary motoneurons, we proceeded to conduct rescue experiments in motoneurons by using lentiviral vectors. The constructs were designed to express 7SK deletion mutants under the mouse U6 promoter and at the same time to drive expression of a 7SK shRNA from an H1 promoter for the depletion of endogenous 7SK. Using this system we found that 7SK mutants harboring deletions of either SL1 or SL3 could not rescue the axon growth defect of 7SK-depleted motoneurons suggesting that 7SK/hnRNP R complexes are integral for this process. In order to identify novel 7SK binding proteins and investigate their functions, I proceeded to conduct pull-down experiments by using a biotinylated RNA antisense oligonucleotide that targets the U17-C33 region of 7SK thereby purifying endogenous 7SK complexes. Following mass spectrometry of purified 7SK complexes, we identified a number of novel 7SK interactors. Among these is the Smn complex. Deficiency of the Smn complex causes the motoneuron disease spinal muscular atrophy (SMA) characterized by loss of lower motoneurons in the spinal cord. Smn has previously been shown to interact with hnRNP R. Accordingly, we found Smn as part of 7SK/hnRNP R complexes. These proteomics data suggest that 7SK potentially plays important roles in different signaling pathways in addition to transcription. / Bei Säugetieren wird ein großer Teil des Genoms als nicht-kodierende RNAs transkribiert. Es gibt immer mehr Hinweise darauf, dass nicht-kodierende RNAs eine wichtige Rolle sowohl für die normale Zellfunktion als auch bei Krankheitsprozessen wie Krebs oder Neurodegeneration spielen. Die Interpretation der Funktionen nicht-kodierender RNAs und der molekularen Mechanismen, über die sie wirken, ist eine der wichtigsten Herausforderungen, denen die RNA-Biologie heute gegenübersteht. In meiner Promotionsarbeit habe ich die Rolle von 7SK, einer der am häufigsten vorkommenden nicht-kodierenden RNAs, bei der Entwicklung und Funktion von Motoneuronen untersucht. 7SK ist eine RNA, die aus 331 Nukleotiden besteht und deren Struktur bekannt ist. Sie wird von der RNA-Polymerase III transkribiert. Sie bildet vier Stem-Loop (SL)-Strukturen, die als Bindungsstellen für verschiedene Proteine dienen. LARP7 bindet an SL4 und schützt das 3'-Ende vor exonukleolytischem Abbau. SL1 dient als Bindungsstelle für HEXIM1, das den P-TEFb-Komplex rekrutiert, der aus CDK9 und Cyclin T1 besteht. P-TEFb hat eine stimulierende Rolle für die Transkription und wird durch Sequestrierung durch 7SK reguliert. In jüngerer Zeit wurde eine Reihe von heterogenen nukleären Ribonukleoproteinen (hnRNPs) als 7SK-Interaktoren identifiziert. Eines davon ist hnRNP R, von dem gezeigt wurde, dass es eine Rolle bei der Entwicklung von Motoneuronen spielt, indem es das Axonwachstum reguliert. Durch die Interaktion mit P-TEFb und RNA-bindenden Proteinen kann 7SK als Drehscheibe für die Rekrutierung und Freisetzung verschiedener Proteine betrachtet werden. Die Fragen, mit denen ich mich während meiner Doktorarbeit beschäftigt habe, lauten wie folgt: 1) Welche Region von 7SK interagiert mit hnRNP R, einem Hauptinteraktor von 7SK? 2) Welche Effekte treten in Motoneuronen auf, wenn die Bindung von hnRNP R an 7SK inhibiert wird? 3) Gibt es zusätzliche 7SK-bindende Proteine, die die Funktionen des 7SK RNPs regulieren? Mit Hilfe von in vitro und in vivo Experimenten fand ich heraus, dass hnRNP R sowohl die SL1- als auch die SL3-Region von 7SK bindet, und dass P-TEFb nach der Deletion der SL1-Region nicht rekrutiert werden kann, aber in der Lage ist, an eine 7SK-Mutante mit Deletion von SL3 zu binden. Um die Frage zu beantworten, wie sich die 7SK-Mutationen auf Axonwachstum in primären Motoneuronen der Maus auswirken, führten wir Rettungsexperimente an Motoneuronen unter Verwendung lentiviraler Vektoren durch. Die Konstrukte wurden so konzipiert, dass sie 7SK-Deletionsmutanten durch den U6-Promotor der Maus exprimieren und gleichzeitig eine 7SK-shRNA von einem H1-Promotor für die Depletion von endogenem 7SK transkribieren. Mit diesem System fanden wir heraus, dass 7SK-Mutanten, die Deletionen von SL1 oder SL3 beherbergen, den Axon-Wachstumsdefekt von 7SK-depletierten Motoneuronen nicht retten konnten, was darauf hindeutet, dass 7SK/hnRNP R-Komplexe für diesen Prozess von Bedeutung sind. Um neue 7SK-Bindungsproteine zu identifizieren und ihre Funktionen zu untersuchen, führte ich Pulldown-Experimente durch, bei denen ich ein biotinyliertes RNA-Antisense-Oligonukleotid verwendete, das an die U17-C33-Region von 7SK bindet und dadurch Aufreinigung endogener 7SK-Komplexe erlaubt. Nach der Massenspektrometrie der gereinigten 7SK-Komplexe identifizierten wir eine Reihe neuer 7SK-Interaktoren. Einer davon ist der Smn-Komplex. Ein Mangel des Smn-Komplexes verursacht die Motoneuronerkrankung Spinale Muskelatrophie (SMA), die durch den Verlust der unteren Motoneuronen im Rückenmark gekennzeichnet ist. Es wurde bereits gezeigt, dass Smn mit hnRNP R interagiert. Dementsprechend fanden wir Smn als Teil des 7SK/hnRNP R-Komplexes. Diese Proteom-Daten deuten darauf hin, dass 7SK neben der Transkription möglicherweise auch in anderen Signalwegen wie der spliceosomalen snRNP Biogenese eine wichtige Rolle spielt.

L’ARN non-codant 7SK forme la charpente d’un complexe, 7SK snRNP, qui régule l’activité du facteur d’élongation de la transcription P-TEFb, intervenant dans la levée des pauses transcriptionelles chez les métazoaires. Le 7SK snRNP comprend les protéines LARP7, essentielle pour la stabilité de l’ARN 7SK et MePCE, participant à sa coiffe. Dans le cadre d’une investigation du rapport entre structure et fonction de l’ARN7SK, le projet était de comprendre commen la protéine LARP7 reconnait et assemble l’ARN dans le 7SK snRNP. La protéine LARP7, membre d’une famille reliée à laprotéine La, est spécifique de 7SK. Les éléments responsables de l’interaction ont été analysés par des méthodes biochimiques dans des complexes reconstitués à partir d’ARN synthétique et de protéines recombinantes. Le module La, dans la région N-terminale, reconnaît et lie les trois uridines à l’extrémité 3’ de l’ARN et, additionellement,une séquence conservée au pied de la tigeboucle en 3’, induisant une conformation fermée de l’ARN. L’autre extrémité de la protéine comprend un domain RRM de reconnaissance de l’ARN, qui se lie à la boucle apicale de la tige-boucle 3’. La protéine LARP7 reconnaît également une région conservée au centre de l’ARN. Dans l’ensemble, LARP7 utiliserait ses domaines terminaux et central pour envelopper l’ARN et le stabiliser. Au cours de ces travaux, une interaction directe du domaine C-terminal avec la tige-boucle 5’ a également été mise en évidence. Celle-ci comprend le site de liaison à la HEXIM, la protéine qui déclenche l’interaction avec P-TEFb et un rôle fonctionnel de LARP7 est envisagé. / The non-coding RNA 7SK is the scaffold for the 7SK snRNP complex that regulates PTEFb, the positive transcription elongation factor, which relieves transcription pauses in metazoans. The 7SK snRNP comprises the proteins LARP7, essential for 7SK stability and MePCE, involved in capping. In the frame of an investigation of how the structure of the7SK RNA sustains its function, the project was to understand how is the RNA recognized and assembled in the 7SK snRNPby the associated protein LARP7. LARP7, a La-related protein is specific for 7SK. The elements responsible for the interaction were investigated by biochemical approaches in vitro with complexes reconstituted from purified recombinant proteins and transcribed RNA. The La-module of LARP7 recognizes and binds the triplet of uridines at the 3’-end of the 7SK RNA and additionally binds to a conserved region at the foot of the 3’-hairpin.This may stabilize a closed conformation of the 7SK. On the other end of the LARP7molecule, the C-terminal domain comprising a RRM (RNA Recognition Motif) binds to the apical loop of the 3’hairpin. Further investigations showed that a conserved region in the core of the RNA is also involved. On the whole, this strongly suggests thatLARP7 wraps around 7SK using its N terminal, C-terminal and linker domains to ensure the RNA stabilization into a functional core. In the course of the investigation, was revealed a direct interaction of the C-terminal domain of LARP7 with the 5’-hairpin of the RNA, which is responsible for 7SK function as it contains the binding site of HEXIM, the protein which bridges 7SK and P-TEFb. A possible functional role of LARP7 is envisioned.

The 7SK small nuclear RNA (snRNA) is a key player in the regulation of polymerase (pol) II transcription. The 7SK RNA was long believed to be specific to vertebrates where it is highly conserved. Homologs in basal deuterostomes and a few lophotrochozoan species were only recently reported. On longer timescales, 7SK evolves rapidly with only few conserved sequence and structure motifs. Previous attempts to identify the Drosophila homolog thus have remained unsuccessful despite considerable efforts. Here we report on the discovery of arthropod 7SK RNAs using a novel search strategy based on pol III promoters, as well as the subsequent verification of its expression. Our results demonstrate that a 7SK snRNA featuring 2 highly structured conserved domains was present already in the bilaterian ancestor.

7SK RNA is a highly abundant noncoding RNA in mammalian cells whose function in transcriptional regulation has only recently been elucidated. Despite its highly conserved sequence throughout vertebrates, all attempts to discover 7SK RNA homologues in invertebrate species have failed so far. Here we report on a combined experimental and computational survey that succeeded in discovering 7SK RNAs in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. Despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. The additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3´ and 5´ stemloop motifs, and provide evidence for a third, structurally well-conserved domain.

No description available.

No description available.

The process of protein and RNA folding has been understood in general terms through the principle of minimal frustration, and is usually thought of as being guided by a folding funnel on the energy landscape, which is based around the native structure. However, more recently, various biomolecules have been associated with multifunnel energy landscapes, where each funnel exhibits a distinct structural ensemble and function. This work explores how the principle of minimal frustration may be extended to multifunnel energy landscapes that are associated with multifunctional biomolecules. To achieve this aim, the computational potential energy landscape framework is employed to analyse four example systems. Additionally, this study analyses mutants for all four systems, where the mutations are chosen to change properties of the systems without destabilising the native sequence ensemble entirely. The first system considered is a two-state coiled-coil. It is shown how mutations fundamentally change the energy landscape from the minimal frustrated organisation necessary to fulfil biological function. These changes can introduce alternative pathways for folding, as well as new structural ensembles. Similar effects are observed for ubiquitin. In addition, the landscape exploration allows us to calculate a number of experimentally determined properties for this protein, which exhibit excellent agreement, and we characterise folding at an atomistic level of detail. Next we consider the hormones oxytocin and vasopressin, which are themselves mutants of each other, along with a number of other mutants for both molecules. Again, the frustration in the landscape increases due to mutations, and a greater variety in the resulting structural ensembles is observed, leading to changes in binding affinities. Finally, the HP1 loop of RNA 7SK is analysed, revealing that the principles established for the energy landscapes of proteins extend to nucleic acids. Overall, the results indicate that sequences have evolved to exhibit the minimum number of funnels on the energy landscape to support multiple functions, extending the principle of minimal frustration to multifunnel energy landscapes.