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The human CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP) binds to the G-rich elements in target mRNA coding sequences and promotes translation / Das humane CCHC-Typ-Zinkfinger-Nukleinsäure-Binde-Protein (CNBP) bindet an G-reiche Elemente in der kodierenden Sequenz seiner Ziel-mRNAs und fördert deren TranslationGupta, Sanjay Kumar January 2017 (has links) (PDF)
The genetic information encoded with in the genes are transcribed and translated to give rise to
the functional proteins, which are building block of a cell. At first, it was thought that the
regulation of gene expression particularly occurs at the level of transcription by various
transcription factors. Recent discoveries have shown the vital role of gene regulation at the level
of RNA also known as post-transcriptional gene regulation (PTGR). Apart from non-coding RNAs
e.g. micro RNAs, various RNA binding proteins (RBPs) play essential role in PTGR. RBPs have
been implicated in different stages of mRNA life cycle ranging from splicing, processing,
transport, localization and decay. In last 20 years studies have shown the presence of hundreds
of RBPs across eukaryotic systems many of which are widely conserved. Given the rising number
of RBPs and their link to human diseases it is quite evident that RBPs have major role in cellular
processes and their regulation. The current study is aimed to describe the so far unknown
molecular mechanism of CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP/ZNF9)
function in vivo.
CNBP is ubiquitously expressed across various human tissues and is a highly conserved RBP in
eukaryotes. It is required for embryonic development in mammals and has been implicated in
transcriptional as well as post-transcriptional gene regulation; however, its molecular function
and direct target genes remain elusive. Here, we use multiple systems-wide approaches to
identify CNBP targets and document the consequences of CNBP binding. We established CNBP as
a cytoplasmic RNA-binding-protein and used Photoactivatable Ribonucleoside Enhanced
Crosslinking and Immunoprecipitation (PAR-CLIP) to identify direct interactions of CNBP with
4178 mRNAs. CNBP preferentially bound a G-rich motif in the target mRNA coding sequences.
Functional analyses, including ribosome profiling, RNA sequencing, and luciferase assays
revealed the CNBP mode of action on target transcripts. CNBP binding was found to increase the
translational efficiency of its target genes. We hypothesize that this is consistent with an RNA
chaperone function of CNBP helping to resolve secondary structures, thus promoting
translation. Altogether this study provides a novel mechanism of CNBP function in vivo and acts
as a step-stone to study the individual CNBP targets that will bring us closer to understand the
disease onset. / Die in der DNA kodierte genetische Information wird transkribiert und
translatiert, um funktionelle Proteine zu bilden, welche die Bausteine von Zellen sind. Lange Zeit wurde vermutet, dass die Regulation der Genexpression insbesondere auf dem Level der Transkription erfolgt. Kürzlich gemachte Entdeckungen haben jedoch die zentrale Rolle der Genregulation auf dem
Level der RNA, auch bekannt als posttranskriptionelle Genregulation (PTGR), gezeigt. Neben
nicht-kodierenden RNAs wie microRNAs, besitzen verschiedene RNA-Binde-Proteine (RBP) eine
Schlüsselrolle in der PTGR. RBPs wurden mit diversen Ebenen des mRNA- Lebenszyklus, wie Speißen,
Prozessieren, Transport, Lokalisation und Abbau in Verbindung gebracht. In den letzten 20 Jahren
haben Studien die Existenz von Hunderten von RBPs in unterschiedlichen eukaryotischen Systemen
gezeigt, von denen viele weithin konserviert sind. Bedenkt man die steigende Anzahl entdeckter und
charakterisierter RBPs und ihren Bezug zu Krankheiten des Menschen, so ist es offensichtlich, dass
RBPs eine große Rolle in der Regulation zellulärer Prozesse besitzen. Das Ziel der hier
vorliegenden Studie bestand darin, die bis jetzt unbekannten molekularen Mechanismen der Funktion
des CCHC-Typ-Zinkfinger-Nukleinsäure- Binde-Proteins (CNBP/ZNF9) in vivo zu beschreiben.
CNBP ist in verschiedenen humanen Geweben ubiquitär exprimiert und ein hoch konserviertes RBP in
Eukaryoten. Es ist für die embryonale Entwicklung in Säugetieren notwendig und wurde mit der
transkriptionellen und posttranskriptionellen Genregulation in Verbindung gebracht. Seine
molekulare Funktion sowie die unmittelbaren Zielgene blieben jedoch unklar. In dieser Studie
verwendeten wir systemweit analysierende Methoden um CNPB-Zieltranskripte zu identifizieren und
dokumentierten die Folgen der Bindung von CNBP an diese. Wir haben CNBP als ein zytoplasmatisches
RNA-Binde-Protein charakterisiert und Quervernetzung und Immunpräzipitation mit photoaktivierbaren
Ribonukleotiden (PAR-CLIP) angewendet. Dabei wurden direkte Interaktionen von CNBP mit 4178 mRNAs
identifiziert. CNBP bindet bevorzugt an ein G-reiches Motiv in der kodierenden Sequenz der
Ziel-mRNA. Funktionale Analysen, unter anderem Ribosom-Profil-Untersuchungen, RNA Sequenzierung
und Luciferaseproben, zeigten die Art und Weise, wie CNBP auf die Zieltranskripte wirkt. Die
Bindung von CNBP an seine Zieltranskripte erhöht deren Translationseffizienz. Wir vermuten, dass
dies eine RNA-Chaperon- Funktion von CNBP darstellt, die hilft Sekundärstrukturen aufzulösen und
die Translation zu fördern. Zusammengefasst liefert diese Studie einen neuen Mechanismus der
Funktion von CNBP in vivo und kann als Startpunkt dienen um einzelne CNBP Ziele zu untersuchen.
Dies wird
uns helfen dem Verständnis der Krankheitsentstehung näher zu kommen. ...
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THE CELLULAR NUCLEIC ACID BINDING PROTEIN REGULATES THE ALZHEIMER’S DISEASE β-SECRETASE PROTEIN BACE1Holler, Christopher J 01 January 2012 (has links)
Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly population and is believed to be caused by the overproduction and accumulation of the toxic amyloid beta (Aβ) peptide in the brain. Aβ is produced by two separate enzymatic cleavage events of the larger membrane bound amyloid precursor protein, APP. The first, and rate-limiting, cleavage event is made by beta-secretase, or BACE1, and is thus an attractive therapeutic target. Our lab, as well as many others, has shown that BACE1 protein and activity are increased in late-stage sporadic AD. We have extended these findings to show that BACE1 is increased in the earliest stages of AD before the onset of significant Aβ accumulation, indicating a potential causal role in the disease. Interestingly, BACE1 mRNA levels are unchanged in AD, leading to reason that a post-transcriptional method of BACE1 regulation is altered in disease. To date, the mechanism for this aberrant post-transcriptional regulation has not been elucidated. This study has implicated the cellular nucleic acid binding protein (CNBP), a highly conserved RNA binding protein, as a positive regulator of BACE1 translation, with implications for the etiology of sporadic AD. CNBP overexpression in cultured cells or spiked into a cell-free in vitro translation system increased BACE1 protein expression without affecting BACE1 mRNA levels. Knockdown of CNBP reduced BACE1 protein and mRNA slightly. Furthermore, CNBP associated with BACE1 mRNA in cell lysates and bound directly to the BACE1 5’ UTR in vitro, which confers most of the regulatory activity. Importantly, CNBP was increased in the progression of AD and correlated with BACE1 expression. Cellular stressors (such as glucose deprivation and oxidative stress) that occur in the AD brain increase BACE1 translation and we have found that these stressors increased CNBP expression as well. Early experimental evidence suggests that CNBP may enhance BACE1 translation through a cap-independent mechanism, which is an alternative translational pathway activated by cell stress. These studies indicate that the RNA binding protein CNBP is a novel trans-acting factor important for the regulation of BACE1 protein production and may be a viable therapeutic target for AD.
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