Post-transcriptional Regulation through Long Noncoding RNAs (lncRNAs)

This book is a collection of eight articles, of which seven are reviews and one is a research paper, that together form a Special Issue that describes the roles that long noncoding RNAs (lncRNA) play in gene regulation at a post-transcriptional level

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.An interesting feature of Bcl-xL protein is the presence of an unstructured loop domain between its α1 and α2 helices, a domain not essential for its anti-apoptotic function and absent in CED-9, ortholog protein in Caenorhabditis elegans. Within this domain, Bcl-xL undergoes dynamic phosphorylation and dephosphorylation at Ser49 and Ser62 during G2 and mitosis in human cancer cells. When these residues are mutated and proteins expressed in cancer cells, cells harbor mitotic defects, including chromosome mis-attachment, lagging, bridging and mis-segregation, events associated with chromosome instability and aneuploidy. To further analyze the effects of Bcl-xL Ser49 and Ser62 in normal cells, the present studies were performed in normal human diploid cells, and in vivo in Caenorhabditis elegans. First, we studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL(wild type), (S49A), (S49D), (S62A), (S62D) and the dual (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL Ser49 and Ser62 phosphorylation/ dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. Second, we undertook experiments in Caenorhabditis elegans to understand the importance of Bcl-xL Ser49 and Ser62 in vivo. Transgenic worms carrying single-site S49A, S62A, S49D, S62D and dual-site S49/62A mutants were generated and their effects were analyzed in germlines of young adult worms. Worms expressing Bcl-xL variants showed decreased egg-laying and hatching, variations in the length of their mitotic regions and transition zones, chromosomal abnormalities at their diplotene stages, and increased germline apoptosis. Some of these transgenic strains, particularly the Ser to Ala variants, also showed slight modulations of lifespan compared to their controls. The in vivo observations confirmed the importance of Ser49 and Ser62 within the loop domain of Bcl-xL in maintaining chromosome stability. These studies could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy

Insights into the regulation of aging

Aging is doubtlessly one of the most complex and multi-factorial biological processes we have encountered since the beginning of modern life sciences and the systematic study of human and animal biology. Despite many remarkable findings, aging remains an incompletely understood mechanism, causing several severe diseases, such as cardiovascular diseases, neurodegenerative diseases or cancer. It is associated with a progressive loss of cell functions that lead to a decline of tissue functions and finally resulting in death. Uncountable studies were performed over the last five decades to identify possible causes of how and why we age. Nevertheless, there is a still ongoing debate about the true molecular source of aging, giving rise to a variety of competing theories. Due to its highly complex nature, we have investigated aging from various perspectives, based on the gene expression of different species and tissues. We analyzed a huge set of RNA-Seq transcriptomic data to obtain new insights into the genetic regulation of aging and to identify conserved molecular processes that might be responsible for aging-related disorders. We found that each tissue shows its own distinct pattern of gene expressional changes with age, because they have to respond to different types of stress over time, leading to differing sources of molecular damage and subsequent stress responses. In particular, we could show this for four wellstudied aging-related processes: cellular senescence, inflammation, oxidative stress response and circadian rhythms. In addition, we could show that alternative splicing (i.e., the generation of multiple mRNA isoforms from single genes) is in general only slightly affected by aging and probably plays a secondary role in the overall aging process. In contrast, we found microRNAs (very small regulatory RNA molecules) to be important modulators of aging in all investigated pecies and tissues. Concluding, the results presented in this thesis describe aging as a stochastic process, leading to an accumulation of different kinds of molecular damage and the respective cellular stress responses. We have identified several genetic factors that could serve as potential diagnostic markers or even therapeutic targets, that could help in the future to slow down the progression of age-associated disorders or preventing them. Nevertheless, the subject of aging remains a challenging research field and many open questions still wait to be answered.Das Altern ist zweifellos einer der komplexesten und faktorenreichsten biologischen Prozesse, dem wir seit Beginn der modernen Lebenswissenschaften und der systematischen Erforschung der Human- und Tierbiologie begegnet sind. Trotz vieler bemerkenswerter Erkenntnisse bleibt das Altern ein unvollständig verstandener Mechanismus, der mit vielen schweren Krankheiten assoziert ist, wie etwa Herz-Kreislauf-Erkrankungen, neurodegenerative Erkrankungen oder Krebs. Es geht mit einem fortschreitenden Verlust von Zellfunktionen einher, der zu einer Abnahme der Organfunktionen und schließlich zum Tod führt. In den letzten fünf Jahrzehnten wurden unzählige Studien durchgeführt, um mögliche Quellen zu finden, wie und warum wir altern. Auch heutzutage wird noch intensiv über die eigentliche molekulare Ursache des Alterns gestritten, was zu einer Vielzahl konkurrierender Theorien führt. Aufgrund seiner hohen Komplexität haben wir in der vorliegenden Arbeit das Altern aus verschiedenen Perspektiven untersucht, basierend auf der Genexpression verschiedener Spezies und Gewebstypen. Wir haben eine Vielzahl an RNA-Seq Transkriptomdaten analysiert, um neue Einblicke in die genetische Regulation des Alterns zu erhalten und um konservierte molekulare Prozesse zu identifizieren, die möglicherweise für altersbedingte Gebrechen und Krankheiten verantwortlich sind. Wir fanden heraus, dass jedes Gewebe mit dem Alter ein eigenes Muster von Genexpressionsänderungen aufweist, da es im Laufe der Zeit auf verschiedene Arten von Stress reagieren muss, was zu unterschiedlichen Ursachen für molekulare Schäden und nachfolgende Stressreaktionen führt. Insbesondere konnten wir dies für vier gut untersuchte, altersbedingte Prozesse zeigen: zelluläre Seneszenz, Entzündungsreaktionen, oxidative Stressreaktion und zirkadiane Rhythmen. Außerdem konnte gezeigt werden, dass alternatives Spleißen (d. H., die Erzeugung mehrerer mRNAIsoformen aus einzelnen Genen) im Allgemeinen nur geringfügig vom Altern betroffen ist und wahrscheinlich eine untergeordnete Rolle im gesamten Alterungsprozess spielt. Im Gegensatz dazu haben wir festgestellt, dass microRNAs (sehr kleine regulatorische RNA-Moleküle) in allen untersuchten Spezies und Geweben wichtige Alterungsmodulatoren sind. Abschließend beschreiben die in dieser Arbeit vorgestellten Ergebnisse das Altern als einen stochastischen Prozess, der zu einer Anhäufung verschiedener Arten von molekularen Schäden und den jeweiligen zellulären Stressreaktionen führt. Wir haben mehrere genetische Faktoren identifiziert, die als potenzielle diagnostische Marker oder sogar als therapeutische Ziele dienen könnten, die in Zukunft dazu beitragen könnten, das Fortschreiten altersbedingter Erkrankungen zu verlangsamen oder zu verhindern. Trotzdem bleibt das Thema Altern weiterhin ein herausforderndes Forschungsfeld und viele offene Fragen warten noch auf ihre Beantwortung

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Insights into the interplay between sumoylation of chromatin-associated proteins and DNA

The maintenance of correct genome sequence is an essential cellular process in which the small ubiquitin-like modifier SUMO plays an important role, probably because its post-translational conjugation to certain proteins can regulate DNA metabolism. The identities of these proteins remain largely unknown, as do how their sumoylation is controlled or what effects their modification has. I was therefore interested in identifying such factors and studying the upstream signals and downstream consequences of their sumoylation, with a particular focus on how DNA may be involved in these processes. To address this problem, I initially used Xenopus laevis egg extracts to isolate and identify SUMO conjugates from replicating chromatin, as it should be enriched for proteins involved in DNA metabolism. I found that progression through S phase, but not genotoxic stress, altered the abundance of chromatin-associated SUMO conjugates. A proteomic analysis of these species during unperturbed and disrupted S phase identified several proteins with a role in DNA metabolism as putative sumoylation substrates. Some of these modification events were confirmed by western blotting and were also shown to be conserved in the budding yeast Saccharomyces cerevisiae. By further investigating the modification of one of the candidates I discovered, i.e. ORC1, the largest subunit of the six-membered origin recognition complex (ORC), I found that all of the ORC subunits were sumoylated. I, however, also observed that manipulating the general levels of sumoylation in either egg extracts or budding yeast did not affect the recognized functions of ORC in DNA replication, thus indicating that sumoylation does not play a significant role in such a process. I therefore focused on the sumoylation of another candidate I found in my screen, the DNA-break sensor poly(ADP-Ribose) polymerase 1, PARP-1. In vitro, I found that sumoylation of both the full-length protein and its DNA-binding domain alone depended on the presence of intact DNA and was strongly inhibited by nicks in the double helix. In vivo, two main sites of sumoylation in PARP-1 were identified. By mutating them and creating a linear PARP-1-SUMO fusion, to mimic a constitutively sumoylated polymerase, I investigated the functions of PARP-1 sumoylation in human cells. I found that the sumoylation of PARP-1 did not affect the protein’s catalytic activity, localization or binding to intact chromatin or nicked DNA. Instead, sumoylation appeared to accelerate PARP-1’s ubiquitylation and subsequent degradation. These observations exemplify how DNA and sumoylation can interplay with each other to control the properties of chromatin-associated proteins. They also suggest that when PARP-1 is associated to single-stranded DNA breaks, and is therefore engaged in DNA repair, it becomes refractory to sumoylation and subsequent degradation. Thus, sumoylation may help cells to distinguish between two functionally distinct subpopulations of PARP-1: one that is not sumoylated because it is bound to, and in the process of repairing, DNA nicks, and one that is sumoylated because it is bound to intact DNA, which could be involved in other processes in which the polymerase plays a role, such as transcription regulation