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Title
Identification and characterization of the cold-inducible RNA-binding protein CIRP as a regulator of p27Kip1 translation / Mag. Biol. Martina Roilo
AuthorRoilo, Martina
CensorDoppler, Wolfgang ; Geley, Stephan
Thesis advisorHengst, Ludger
PublishedInnsbruck, April 2018
Description155 Blätter : Illustrationen, Diagramme
Institutional NoteMedical University Innsbruck, Dissertation, 2018
Date of SubmissionMay 2018
LanguageEnglish
Document typeDissertation (PhD)
Keywords (DE)RNA-bindendes Protein / Translation / p27 / Milde Hypothermie / Stress Granules
Keywords (EN)RNA-binding protein / Translation / p27 / Mild hypothermia / Stress granules
URNurn:nbn:at:at-ubi:1-31712 Persistent Identifier (URN)
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 The work is publicly available
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Identification and characterization of the cold-inducible RNA-binding protein CIRP as a regulator of p27Kip1 translation [26.89 mb]
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Abstract (German)

p27Kip1 ist ein CDK-Inhibitor Protein, welches eine zentrale Rolle in der Kontrolle der Zellproliferation und des Zellzyklus spielt. p27 wird in differenzierenden und nicht-proliferierenden Zellen stark exprimiert, wogegen mitogene Faktoren die Expression von p27 über verschiedene Signalwege unterdrücken. In proliferierenden Zellen ist p27 in G0 und der frühen G1 Phase exprimiert; vor dem Beginn der S Phase wird das Protein abgebaut. Während die Transkription von p27 im Zellzyklus meistens nicht reguliert ist, kann die translationale Regulation entscheidend sein, um die Expression von p27 vor dem “Restriction point” in der späten G1 Phase zu bestimmen und dadurch die Zellzyklusprogression zu kontrollieren.

Um die Mechanismen, die die Expression und insbesondere die Translation von p27 kontrollieren, zu untersuchen, wurde in unserem Labor ein “Northwestern screen” durchgeführt, wodurch potentielle neue p27 5UTR-bindende Proteine identifiziert werden konnten. Diese könnten neue Wege der p27 Translation aufdecken. Eines dieser Proteine war das Cold-Inducible RNA-binding Protein (CIRP). CIRP ist ein Protein, das in unterschiedlichen Stressbedingungen induziert und/oder anders lokalisiert werden kann. Wir haben entdeckt, dass CIRP die Translation von p27 über die 5UTR des Transkriptes stimulieren kann. CIRP kann möglicherweise die Aktivität von p27 IRES erhöhen, jedoch sind die genaue molekulare Mechanismen der IRES-abhängigen Translation und deren Regulation durch interagierenden Proteinen noch zu klären. Wir vermuten, dass die Assemblierung eines nukleären Protein-mRNA Komplexes für die Induktion der p27 Translation durch CIRP erforderlich sein könnte.

Wie der Name des Proteins schon sagt, wird CIRP in Zellen unter milden Hypothermiebedingugen (32C) induziert. Unter solchen Bedingungen konnten wir eine erhöhte Translation von p27 nachweisen. Es ist bekannt, dass milde Hypothermie zu einer Verringerung der metabolischen Aktivität und zu einer Abnahme der Zellproliferation führt. Wir konnten nun zeigen, dass p27 für diese verringerte Proliferation von zentraler Bedeutung ist. In Abwesenheit von p27 ist die Proliferationsrate in Zellen unter Kältestress kaum betroffen, während sie in Kontrollzellen unter Kältestress signifikant verzögert ist. Dies deutet darauf hin, dass die CIRP-abhängige Induktion von p27 unter milder Hypothermie die Hemmung der Zellproliferation auslösen könnte.

Darüberhinaus haben wir beobachtet, dass CIRP mit der p27 mRNA in Natriumarsenit-induzierten Stress Granules kolokalisiert; unter diesen Stressbedingungen wird die Expression von p27 Protein reduziert. Wir vermuten, dass durch die Relokalisierung von der p27 mRNA in Stress Granules die Proteintranslation selektiv unterdrückt werden könnte und möglicherweise die p27 mRNA vor Degradation geschützt werden könnte. Weitere Experimente sollten diese Hypothese prüfen und bestimmen, ob CIRP oder andere RNA-bindende Proteine in diesem Prozess eine Rolle spielen.

Insgesamt enthüllt diese Arbeit neue Pathways für die stressabhängige Regulation des Zellzyklusinhibitors p27, durch den das Zellzyklus gezielt gesteuert werden kann.

Abstract (English)

p27Kip1 is a cyclin-dependent kinase inhibitor that plays a fundamental role in controlling cell cycle progression and cell proliferation. p27Kip1 protein accumulates in differentiating cells and upon anti-proliferative signalling, whereas mitogenic stimuli suppress p27Kip1 by multiple pathways. In proliferating cells, p27Kip1 is expressed at high levels in G0 and early G1 phases of the cell cycle, and it decreases prior to S phase entry mainly as a consequence of increased protein degradation. p27Kip1 transcription is frequently not regulated during the cell cycle. p27Kip1 translational control is still poorly understood, but an enhanced p27Kip1 translation rate can promote quiescence or cell differentiation. Regulation of protein translation might contribute to the determination of p27Kip1 protein levels and cell cycle progression prior to restriction point passage.

In order to better understand the mechanisms controlling the expression of p27Kip1, we sought to identify novel p27Kip1 mRNA 5UTR binding proteins that might uncover unknown pathways regulating p27Kip1 translation. By applying a “Northwestern” procedure to protein expression arrays, we identified the cold-inducible RNA-binding protein (CIRP) as one of the protein clones that was able to bind p27Kip1 5UTR. CIRP is a stress responsive protein that can be induced or relocalized in response to a variety of stress stimuli. In this study, we have uncovered CIRP as positive regulator of p27Kip1. CIRP can stimulate p27Kip1 translation by regulating the 5UTR of p27Kip1 mRNA. The existence of an IRES in the p27Kip1 mRNA has been a matter of debate; in support to previous findings, we confirmed that a region of the p27Kip1 5UTR enables cap-independent translation initiation and propose that CIRP might further stimulate the activity of p27Kip1 IRES. The exact mechanism controlling p27Kip1 IRES-mediated translation and its potential regulation by CIRP remains to be determined; we hypothesize that the assembly of a nuclear protein-mRNA complex may be required for the induction of p27Kip1 translation by CIRP.

CIRP is one of the few known cold-responsive proteins. Its induction has been reported to have different effects on cell proliferation, based on the cell types and environmental and cellular conditions. We discovered that the induction of CIRP in MEFs and HEK293 cells grown in mild hypothermic conditions correlates with increased p27Kip1 protein amounts. This p27Kip1 upregulation occurs at the translational level and is mediated by the 5UTR of the transcript. Knockdown of CIRP could prevent this regulation, suggesting that CIRP is, at least in part, responsible for the induction of p27Kip1 translation at 32C. The enhanced expression of p27Kip1 might influence cell cycle progression and proliferation of cells subjected to mild cold shock. Indeed, while wild type cells significantly reduced their proliferation rate at 32C, the doubling time of p27Kip1 knockout cells was not significantly affected by the temperature reduction. Thus, we propose a novel biological pathway contributing to the cold-induced inhibition of cell proliferation, in which CIRP promotes the translation of p27Kip1, leading to delayed cell cycle progression.

We also investigated the effects of other stress conditions, that might modulate the expression or localization of CIRP, on p27Kip1 protein and mRNA. We observed that CIRP colocalizes with p27Kip1 mRNA in sodium arsenite-induced stress granules; upon arsenite-induced cellular stress, p27Kip1 expression is reduced. We speculate that the relocalization of the p27Kip1 transcript into stress granules might be a mechanism to selectively repress protein translation and possibly protect p27Kip1 mRNA from decay. Additional experiments should be done in the future to investigate this hypothesis and determine the potential role of CIRP or other RNA-binding proteins in this process.

Altogether, the data presented here unveil novel pathways of stress-dependent modulation of the cell cycle inhibitor p27Kip1, and provide a novel direct link between stress response signalling and the cell cycle control machinery.

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