Computational Identification of Four Spliceosomal snRNAs from the Deep-Branching Eukaryote Giardia intestinalis

Funding: Marsden Fund New Zealand Allan Wilson Centre The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.RNAs processing other RNAs is very general in eukaryotes, but is not clear to what extent it is ancestral to eukaryotes. Here we focus on pre-mRNA splicing, one of the most important RNA-processing mechanisms in eukaryotes. In most eukaryotes splicing is predominantly catalysed by the major spliceosome complex, which consists of five uridine-rich small nuclear RNAs (U-snRNAs) and over 200 proteins in humans. Three major spliceosomal introns have been found experimentally in Giardia; one Giardia U-snRNA (U5) and a number of spliceosomal proteins have also been identified. However, because of the low sequence similarity between the Giardia ncRNAs and those of other eukaryotes, the other U-snRNAs of Giardia had not been found. Using two computational methods, candidates for Giardia U1, U2, U4 and U6 snRNAs were identified in this study and shown by RT-PCR to be expressed. We found that identifying a U2 candidate helped identify U6 and U4 based on interactions between them. Secondary structural modelling of the Giardia U-snRNA candidates revealed typical features of eukaryotic U-snRNAs. We demonstrate a successful approach to combine computational and experimental methods to identify expected ncRNAs in a highly divergent protist genome. Our findings reinforce the conclusion that spliceosomal small-nuclear RNAs existed in the last common ancestor of eukaryotes

High Throughput Genome-Wide Survey of Small RNAs from the Parasitic Protists Giardia intestinalis and Trichomonas vaginalis

RNA interference (RNAi) is a set of mechanisms which regulate gene expression in eukaryotes. Key elements of RNAi are small sense and antisense RNAs from 19 to 26 nt generated from double-stranded RNAs. MicroRNAs (miRNAs) are a major type of RNAi-associated small RNAs and are found in most eukaryotes studied to date. To investigate whether small RNAs associated with RNAi appear to be present in all eukaryotic lineages, and therefore present in the ancestral eukaryote, we studied two deep-branching protozoan parasites, Giardia intestinalis and Trichomonas vaginalis. Little is known about endogenous small RNAs involved in RNAi of these organisms. Using Illumina Solexa sequencing and genome-wide analysis of small RNAs from these distantly related deep-branching eukaryotes, we identified 10 strong miRNA candidates from Giardia and 11 from Trichomonas. We also found evidence of Giardia short-interfering RNAs potentially involved in the expression of variant-specific surface proteins. In addition, eight new small nucleolar RNAs from Trichomonas are identified. Our results indicate that miRNAs are likely to be general in ancestral eukaryotes and therefore are likely to be a universal feature of eukaryotes

Ancestral RNA: The RNA biology of the eukaryotic ancestor

Our knowledge of RNA biology within eukaryotes has exploded over the last five years. Within new research we see that some features that were once thought to be part of multicellular life have now been identified in several protist lineages. Hence, it is timely to ask which features of eukaryote RNA biology are ancestral to all eukaryotes. We focus on RNA-based regulation and epigenetic mechanisms that use small regulatory ncRNAs and long ncRNAs, to highlight some of the many questions surrounding eukaryotic ncRNA evolution

Combined experimental and computational approach to identify non-protein-coding RNAs in the deep-branching eukaryote Giardia intestinalis

Non-protein-coding RNAs represent a large proportion of transcribed sequences in eukaryotes. These RNAs often function in large RNA–protein complexes, which are catalysts in various RNA-processing pathways. As RNA processing has become an increasingly important area of research, numerous non-messenger RNAs have been uncovered in all the model eukaryotic organisms. However, knowledge on RNA processing in deep-branching eukaryotes is still limited. This study focuses on the identification of non-protein-coding RNAs from the diplomonad parasite Giardia intestinalis, showing that a combined experimental and computational search strategy is a fast method of screening reduced or compact genomes. The analysis of our Giardia cDNA library has uncovered 31 novel candidates, including C/D-box and H/ACA box snoRNAs, as well as an unusual transcript of RNase P, and double-stranded RNAs. Subsequent computational analysis has revealed additional putative C/D-box snoRNAs. Our results will lead towards a future understanding of RNA metabolism in the deep-branching eukaryote Giardia, as more ncRNAs are characterized

Computational identification of new structured cis-regulatory elements in the 3'-untranslated region of human protein coding genes

Messenger ribonucleic acids (RNAs) contain a large number of cis-regulatory RNA elements that function in many types of post-transcriptional regulation. These cis-regulatory elements are often characterized by conserved structures and/or sequences. Although some classes are well known, given the wide range of RNA-interacting proteins in eukaryotes, it is likely that many new classes of cis-regulatory elements are yet to be discovered. An approach to this is to use computational methods that have the advantage of analysing genomic data, particularly comparative data on a large scale. In this study, a set of structural discovery algorithms was applied followed by support vector machine (SVM) classification. We trained a new classification model (CisRNA-SVM) on a set of known structured cis-regulatory elements from 3′-untranslated regions (UTRs) and successfully distinguished these and groups of cis-regulatory elements not been strained on from control genomic and shuffled sequences. The new method outperformed previous methods in classification of cis-regulatory RNA elements. This model was then used to predict new elements from cross-species conserved regions of human 3′-UTRs. Clustering of these elements identified new classes of potential cis-regulatory elements. The model, training and testing sets and novel human predictions are available at: http://mRNA.otago.ac.nz/CisRNA-SVM

Aspects of room acoustics, vision and motion in the human auditory perception of space

The human sense of hearing contributes to the awareness of where sound-generating objects are located in space and of the environment in which the hearing individual is located. This auditory perception of space interacts in complex ways with our other senses, can be both disrupted and enhanced by sound reflections, and includes safety mechanisms which have evolved to protect our lives, but can also mislead us. This dissertation explores some selected topics from this wide subject area, mostly by testing the abilities and subjective judgments of human listeners in virtual environments. Reverberation is the gradually decaying persistence of sounds in an enclosed space which results from repeated sound reflections at surfaces. The first experiment (Chapter 2) compared how strongly people perceived reverberation in different visual situations: when they could see the room and the source which generated the sound; when they could see some room and some sound source, but the image did not match what they heard; and when they could not see anything at all. There were no indications that the visual image had any influence on this aspect of room-acoustical perception. The potential benefits of motion for judging the distance of sound sources were the focus of the second study (Chapter 3), which consists of two parts. In the first part, loudspeakers were placed at different depths in front of sitting listeners who, on command, had to either remain still or move their upper bodies sideways. This experiment demonstrated that humans can exploit motion parallax (the effect that closer objects appear faster to a moving observer than farther objects) with their ears and not just with their eyes. The second part combined a virtualisation of such sound sources with a motion platform to show that the listeners’ interpretation of this auditory motion parallax was better when they performed this lateral movement by themselves, rather than when they were moved by the apparatus or were not actually in motion at all. Two more experiments were concerned with the perception of sounds which are perceived as becoming louder over time. These have been called “looming”, as the source of such a sound might be on a collision course. One of the studies (Chapter 4) showed that western diamondback rattlesnakes (Crotalus atrox) increase the vibration speed of their rattle in response to the approach of a threatening object. It also demonstrated that human listeners perceive (virtual) snakes which engage in this behaviour as especially close, causing them to keep a greater margin of safety than they would otherwise. The other study (section 5.6) was concerned with the well-known looming bias of the sound localisation system, a phenomenon which leads to a sometimes exaggerated, sometimes more accurate perception of approaching compared to receding sounds. It attempted to find out whether this bias is affected by whether listeners hear such sounds in a virtual enclosed space or in an environment with no sound reflections. While the results were inconclusive, this experiment is noteworthy as a proof of concept: It was the first study to make use of a new real-time room-acoustical simulation system, liveRAZR, which was developed as part of this dissertation (Chapter 5). Finally, while humans have been more often studied for their unique abilities to communicate with each other and bats for their extraordinary capacity to locate objects by sound, this dissertation turns this setting of priorities on its head with the last paper (Chapter 6): Based on recordings of six pale spear-nosed bats (Phyllostomus discolor), it is a survey of the identifiably distinct vocalisations observed in their social interactions, along with a description of the different situations in which they typically occur.Das menschliche Gehör trägt zum Bewusstsein dafür bei, wo sich schallerzeugende Objekte im Raum befinden und wie die Umgebung beschaffen ist, in der sich eine Person aufhält. Diese auditorische Raumwahrnehmung interagiert auf komplexe Art und Weise mit unseren anderen Sinnen, kann von Schallreflektionen sowohl profitieren als auch durch sie behindert werden, und besitzt Mechanismen welche evolutionär entstanden sind, um unser Leben zu schützen, uns aber auch irreführen können. Diese Dissertation befasst sich mit einigen ausgewählten Themen aus diesem weiten Feld und stützt sich dabei meist auf die Testung von Wahrnehmungsfähigkeiten und subjektiver Einschätzungen menschlicher Hörer/-innen in virtueller Realität. Beim ersten Experiment (Kapitel 2) handelte es sich um einen Vergleich zwischen der Wahrnehmung von Nachhall, dem durch wiederholte Reflexionen an Oberflächen hervorgerufenen, sukzessiv abschwellenden Verbleib von Schall in einem umschlossenen Raum, unter verschiedenen visuellen Umständen: wenn die Versuchsperson den Raum und die Schallquelle sehen konnte; wenn sie irgendeinen Raum und irgendeine Schallquelle sehen konnte, dieses Bild aber vom Schalleindruck abwich; und wenn sie gar kein Bild sehen konnte. Dieser Versuch konnte keinen Einfluss eines Seheindrucks auf diesen Aspekt der raumakustischen Wahrnehmung zu Tage fördern. Mögliche Vorteile von Bewegung für die Einschätzung der Entfernung von Schallquellen waren der Schwerpunkt der zweiten Studie (Kapitel 3). Diese bestand aus zwei Teilen, wovon der erste zeigte, dass Hörer/-innen, die ihren Oberkörper relativ zu zwei in unterschiedlichen Abständen vor ihnen aufgestellten Lautsprechern auf Kommando entweder stillhalten oder seitlich bewegen mussten, im letzteren Falle von der Bewegungsparallaxe (dem Effekt, dass sich der nähere Lautsprecher relativ zum sich bewegenden Körper schneller bewegte als der weiter entfernte) profitieren konnten. Der zweite Teil kombinierte eine Simulation solcher Schallquellen mit einer Bewegungsplattform, wodurch gezeigt werden konnte, dass die bewusste Eigenbewegung für die Versuchspersonen hilfreicher war, als durch die Plattform bewegt zu werden oder gar nicht wirklich in Bewegung zu sein. Zwei weitere Versuche gingen auf die Wahrnehmung von Schallen ein, deren Ursprungsort sich nach und nach näher an den/die Hörer/-in heranbewegte. Derartige Schalle werden auch als „looming“ („anbahnend“) bezeichnet, da eine solche Annäherung bei bedrohlichen Signalen nichts Gutes ahnen lässt. Einer dieser Versuche (Kapitel 4) zeigte zunächst, dass Texas-Klapperschlangen (Crotalus atrox) die Vibrationsgeschwindigkeit der Schwanzrassel steigern, wenn sich ein bedrohliches Objekt ihnen nähert. Menschliche Hörer/-innen nahmen (virtuelle) Schlangen, die dieses Verhalten aufweisen, als besonders nahe wahr und hielten einen größeren Sicherheitsabstand ein, als sie es sonst tun würden. Der andere Versuch (Abschnitt 5.6) versuchte festzustellen, ob die wohlbekannte Neigung unserer Schallwahrnehmung, näherkommende Schalle manchmal übertrieben und manchmal genauer einzuschätzen als sich entfernende, durch Schallreflektionen beeinflusst werden kann. Diese Ergebnisse waren unschlüssig, jedoch bestand die Besonderheit dieses Versuchs darin, dass er erstmals ein neues Echtzeitsystem zur Raumakustiksimulation (liveRAZR) nutzte, welches als Teil dieser Dissertation entwickelt wurde (Kapitel 5). Abschließend (Kapitel 6) wird die Schwerpunktsetzung auf den Kopf gestellt, nach der Menschen öfter auf ihre einmaligen Fähigkeiten zur Kommunikation miteinander untersucht werden und Fledermäuse öfter auf ihre außergewöhnliches Geschick, Objekte durch Schall zu orten: Anhand von Aufnahmen von sechs Kleinen Lanzennasen (Phyllostomus discolor) fasst das Kapitel die klar voneinander unterscheidbaren Laute zusammen, die diese Tiere im sozialen Umgang miteinander produzieren, und beschreibt, in welchen Situationen diese Lauttypen typischerweise auftreten

doi:10.1093/nar/gkm474 Combined experimental and computational

approach to identify non-protein-coding RNAs in the deep-branching eukaryote Giardia intestinali