The post-translational methylation of arginine in the glycine arginine rich region of CHO nucleoin

Nucleolin is a nucleolar protein important for ribosome biogenesis. Nucleolin contains a conserved glycine arginine rich (GAR) domain near its carboxy terminus. GAR domains are defined by repeating tri-peptide, (arginine -glycine-glycine (RGG)) motifs. The arginine in the RGG motif is post-translationally modified by dimethylation on one of the two guanido nitrogens. Although arginine methylation was identified over 30 years ago, the function of this modification remains unknown. The GAR domain of nucleolin is important for nucleic acid binding and for nucleolar localization of nucleolin. This dissertation describes investigates possible in vivo interactions between nucleolin and other nucleolar proteins involved in ribosome biogenesis. This dissertation also addresses the possible roles that arginine dimethylation may play in the function of the GAR domain. A GAR truncated hamster nucleolin (DGAR) localizes to the nucleoplasm and not to the nucleoli of CHO cells. This version of nucleolin was subsequently used to probe the in vivo interactions of nucleolin with other non-ribosomal nucleolar proteins. In support of previous work, DGAR caused endogenous B23 to redistribute to the nucleoplasm, suggesting an in vivo interaction. Endogenous Nopp140 shows no redistribution in the presence of DGAR. The effect of DGAR on the nucleolar protein, fibrillarin, remains unclear. A redistribution of exogenously expressed wild type nucleolin from nucleoli to the nucleoplasm is observed when CHO cells are treated with the methyltransferase inhibitors 5-methyl 5¢ deoxythioadenosine (MTA) and 3-deaza-adenosine (DAA). This redistribution of nucleolin is reversible and independent of protein synthesis. The arginines in the RGG motifs of the GAR domain of hamster nucleolin were changed to lysine, and the fully substituted protein was not a substrate for the RGG specific protein arginine methyltransferase, Hmt1p. The lysine substituted protein bound nucleic acids and behaved in vivo in a manner indistinguishable from wild type nucleolin. These results indicate that methylation is not necessary for in vitro nucleic acid binding or for in vivo localization of nucleolin, and that the redistribution of nucleolin observed following MTA treatment is likely due to inhibiting the methylation of another nucleolar substrate

5-methylcytosine in RNA: detection, enzymatic formation and biological functions

The nucleobase modification 5-methylcytosine (m5C) is widespread both in DNA and different cellular RNAs. The functions and enzymatic mechanisms of DNA m5C-methylation were extensively studied during the last decades. However, the location, the mechanism of formation and the cellular function(s) of the same modified nucleobase in RNA still remain to be elucidated. The recent development of a bisulfite sequencing approach for efficient m5C localization in various RNA molecules puts ribo-m5C in a highly privileged position as one of the few RNA modifications whose detection is amenable to PCR-based amplification and sequencing methods. Additional progress in the field also includes the characterization of several specific RNA methyltransferase enzymes in various organisms, and the discovery of a new and unexpected link between DNA and RNA m5C-methylation. Numerous putative RNA:m5C-MTases have now been identified and are awaiting characterization, including the identification of their RNA substrates and their related cellular functions. In order to bring these recent exciting developments into perspective, this review provides an ordered overview of the detection methods for RNA methylation, of the biochemistry, enzymology and molecular biology of the corresponding modification enzymes, and discusses perspectives for the emerging biological functions of these enzymes

Williams-Beureni sündroomi kromosoomiregiooni valk WBSCR22 kui ribosoomi biogeneesifaktor

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRibosoomide biogeneesil osaleb ligi 300 valku, millest ligi kolmandik on seotud genoomsete haiguste ja kasvajate tekkega. Williams-Beureni sündroom (WBS) on arenguhäire, mida põhjustab umbes 30 geeni sisaldava regiooni puudumine seitsmendast kromosoomist. WBS patsientidel esineb rida erivaid probleeme – südame-veresoonkonna haigused, sidekoe arenguhäired, neuroloogilised probleemid, spetsiifiline kognitiivne profiil, kasvuprobleemid ja iseloomulikud näojooned. Ümberkorraldused WBS kromosoomiregioonis tekivad madalakoopiaarvuliste kordusjärjestuste esinemise tõttu, kuid hetkel pole teada, kuidas üksikute geenide koopiaarvu muutus põhjustab haigustunnuseid. Arvatakse, et paljud geenid on olulised WBS-i tekkel, mistõttu on vajalik nendelt geenidelt ekspresseeritavate valkude funktsioonide uurimine. Üheks WBS lookusest avalduvaks valguks, mille funktsioon enne antud töö raames sooritatud katsete tegemist oli teadmata, on WBSCR22. WBSCR22 iseloomustamine on oluline ka seetõttu, et tema avaldumistase on tõusnud mitmete kasvajate korral ja on teada, et ta mõjutab kasvajarakkude elulemust ja metastaaside teket. Antud doktoritöö esimese osa eesmärgiks oli WBSCR22 funktsiooni uurimine imetaja rakkudes. Leiti, et WBSCR22 valk asub hajusalt rakutuumas, kuid esineb ka valgu kogunemist tuumakesse – piirkonda, kus toimub ribosomaalse RNA (rRNA) süntees ja algab ribosoomide biogenees. Doktoritöös leiti, et WBSCR22 on oluline rakkude kasvuks, ribosoomi väikese subühiku biogeneesiks ja pre-rRNA protsessinguks. Täpsemalt, WBSCR22 ekspressiooni allareguleerimine põhjustab küpse 18S rRNA viimase eellase, 18S-E pre-rRNA, kuhjumist raku tuumas. See toob kaasa 18S rRNA ja ribosoomi väikese subühiku hulga vähenemise tsütoplasmas, mis võib viia rakkude aeglase jagunemiseni. Doktoritöös leiti, et üheks WBSCR22-ga seonduvaks valguks on TRMT112. TRMT112 paiknemine rakus on määratud WBSCR22 poolt: WBSCR22-TRMT112 kompleks asub raku tuumas ning kompleksi teke on vajalik WBSCR22 stabiilsuseks. TRMT112 hulga vähenemisel suunatakse WBSCR22 lagundamisele. Doktoritöö teises osas analüüsiti retroviiruste Gag valgu poolt indutseeritud viiruslaadsete partiklite (VLP-de) valgulist koostist ning avastati, et nendes VLP-des esineb suur hulk rakulisi valke, sealhulgas ribosoomivalke, kuid nende roll VLP-des ei ole teada.In human cells, almost 300 trans-acting factors are required for the production of ribosomes. Investigating the functions of these proteins is of biomedical importance, as one third of the factors are related to genetic diseases and cancer. Williams-Beuren syndrome is a developmental disorder caused by the contiguous deletion of 26–28 genes from chromosome 7. This genomic disorder is characterized by cardiovascular abnormalities, connective tissue anomalies, a characteristic neurocognitive and behavioural profile, growth delay, and a subtle but distinctive facial dysmorphology. The low copy repeat blocks of the seventh chromosome predispose to genomic rearrangements. However, it is not known how the alterations in the copy number of individual genes contribute to the disease phenotypes. Several genes probably contribute to the phenotype and therefore, it is important to study the cellular functions of the proteins expressed from the WBS locus. The gene encoding for WBSCR22 protein is deleted in WBS, and studying the function of WBSCR22 is also relevant for cancer biology, since several works have demonstrated that WBSCR22 is upregulated in various types of cancers and regulates the survival and metastatic potential of cancer cells. The first part of this dissertation is focused on studying the function of the WBSCR22 protein in mammalian cells, and revealed that WBSCR22 is important for cell growth, ribosome biogenesis and pre-rRNA processing. More precisely, silencing of WBSCR22 expression causes the accumulation of 18S-E pre-rRNA, the final precursor of 18S rRNA, in the cell nucleus, leading to a reduced amount of mature 18S rRNA and ribosome small subunit. In addition, it was found that WBSCR22 interacts with TRMT112 protein and that this interaction is important for the stability of WBSCR22 protein. In conditions where the amount of TRMT112 is limited, WBSCR22 is degraded by the proteasome. The second part of this thesis is focused on analysing the protein content of virus-like particles (VLPs) induced by the expression of murine leukaemia virus (MLV) Gag protein. The results revealed that these VLPs contain various cellular proteins, including ribosomal proteins of both subunits

Telomerase-dependent cell cycle regulation requires NOL1 and TINF2 mRNA degradation by HuR modulates telomere function

치과대학/박사Telomerase is a ribonucleoprotein enzyme that plays a critical role in the maintenance of telomere repeats in most eukaryotic organisms. Although overexpression of telomerase in normal human somatic cells is sufficient to overcome replicative senescence and extend a lifespan, the ability of telomerase to promote tumorigenesis could require additional activities that are independent of its role in telomere extension. Here we identify NOL1 (proliferation-associated nuclear antigen 120) as a TERC-binding protein, which is found in association with catalytically active telomerase. We show that NOL1 binds to cyclin D1 promoter at the TCF binding element and activates its transcription. Moreover, telomerase further enhances expression of cyclin D1 gene by interacting with NOL1 and recruitment to the cyclin D1 promoter, demonstrating a role of telomerase as a modulator of NOL1-dependent transcription in human cancer cells. These data suggest that NOL1 could represent a novel mechanism by which telomerase promotes the prolonged expression of growth-promoting genes critical for the maintenance of tumor survival and cell proliferation. (Chi and Delgado-Olguin 2013) These data suggest that a functional interplay between NOL1 and telomerase plays a critical role in bypassing checkpoint signaling pathways and maintaining cell proliferation capacity, essential properties of telomerase required for cancer progression.ope

The Guardians of the Epigenome - Regulation and Role of Nucleotide Modifications

While from a genetic perspective all cells of an organism are identical, they vary greatly in type and function. Major determinants of cellular diversity are epigenetic alterations, including post-synthetic modifications of nucleic acids. In DNA, the best-studied chemical modification is the methylation of cytosine at carbon C5. 5-methylcytosine (5mC) plays a central role in the regulation of gene expression and has been implicated in a variety of biological processes and diseases. While initially considered as a relatively stable epigenetic mark, a family of proteins named Ten eleven translocation (Tet), were recently shown to catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in an iterative oxidation reaction. These oxidized derivatives have been shown to modulate gene expression and control cellular metabolism. Hence, to avert the formation of aberrant, pathogenic DNA modifications, Tet activity must be precisely regulated. Here, we analyzed the potential of methyl-CpG binding domain (MBD) proteins to control Tet dioxygenase activity in vitro and in vivo. We demonstrate that prior binding of Mecp2 and Mbd2 to DNA protects 5mC from Tet1 mediated oxidation in a concentration dependent manner. The mechanism is based on competitive, sequence unspecific binding to DNA and correlates with nucleic acid coverage and retention time of MBD proteins on DNA. Accordingly, we find increased levels of the Tet oxidation product 5hmC at pericentric heterochromatin in neurons of Mecp2 deficient mice with concomitant reactivation of highly methylated major satellite DNA repeats. Moreover, we find increased expression and retrotransposition of endogenous and engineered long interspersed nuclear elements (LINE1) as potential consequence of unconfined Tet1 activity in human cells. Similar to DNA, RNA contains a variety of post-synthetic modifications that extend their chemical information and properties. One out of < 100 noncanonical ribonucleobases is C5-methylated cytosine, which is present in various types of RNA, including ribosomal RNA (rRNA). Recent studies have shown that Tet proteins do not exclusively hydroxylate methylcytosine in DNA, but also act on 5mC in single-stranded ribonucleic acids. Since modification of rRNA potentially requires the localization of Tet proteins to the nucleolus, the place of rRNA synthesis and ribosome assembly, it is important to understand the general principles of protein targeting to this subnuclear compartment. Therefore, we determined the molecular requirements that are necessary and sufficient for the localization and accumulation of peptides and proteins inside nucleoli of living cells. Our data indicate that peptide units composed of consecutive, positively charged arginines with an isoelectric point ≥ 12.6 meet the chemical conditions for nucleolar localization. Consistent with this, we mapped an arginine-rich region within the low complexity insert of Tet2, which accumulated in nucleoli upon deletion of its regulatory N-terminal domain. Using a pH sensitive dye, we revealed that the nucleolus is relatively acidic. Accordingly, we show that arginine-rich peptides, which carry a net positive charge under these electrochemical conditions, interact with negatively charged RNA in vitro. This interaction in turn, is consistent with the conservation of this nucleolar targeting principle from insects to man. Finally we developed a detailed protocol for the visualization of nucleoli in living cells using fluorescently tagged cell-penetrating peptides (CPP), which allows precise nucleolar localization analysis of various proteins, such as Tet. In summary, our data contribute to understanding the regulation of Tet activity outside and (Tet) protein localization inside of nucleoli

Gene Evolution and Function in Arabidopsis Telomere Biology System

Telomeres protect chromosome ends from being recognized as double-strand breaks, and respond to incomplete end-replication through telomerase-mediated extension. POT1 (Protection of Telomere 1) is a highly conserved protein required for capping chromosome ends and for regulating telomere extension by telomerase. Arabidopsis thaliana encodes three POT1 paralogues: POT1a, POT1b, and POT1c. POT1a functions to maintain telomere length homeostasis by promoting telomerase processivity, while POT1b functions in the DNA damage response. POT1c is derived from a recent duplication of the POT1a locus, but its function is unknown. In this dissertation, I examined the function and evolution of POT1c using genetic and biochemical approach. Unlike pot1a mutants which show defects in telomere maintenance, plants lacking POT1c exhibit no obvious telomere-related or developmental phenotypes. Furthermore, the POT1c gene is not expressed under standard growth conditions. Transposable elements (TE) are embedded in the POT1c promoter region; yet, active silencing is not observed. Although POT1a and the dS17 gene, which was created in the same duplication event that gave rise to POT1c, are highly conserved among A. thaliana accessions, POT1c is not. Comparison of POT1a and POT1c loci from species closely related to A. thaliana and A. lyrata indicates that POT1c initially had a functional promoter that was subsequently disrupted by TE insertion. Together, these studies provide new insights into the fate of newly duplicated genes, and the importance of proper regulation of telomere proteins. In addition to the study of the POT1c locus, I have analyzed a newly identified gene (NOP2A) that is implicated in the control of telomere length set point. NOP2A is a conserved rRNA methyltransferase protein that positively correlates with cell proliferation. Telomere length is variable across eukaryotes, but each species establishes a specific set point that allows full protection for chromosome ends. My research shows that mutation in NOP2A locus leads to shorter, but stable telomere length in the Col-0 accession of A. thaliana. These findings provide strong evidence that additional genes that regulate telomeres remain to be discovered

Investigating the role of RNA 5- methylcytosine on ribosomal and transfer RNAs in Arabidopsis thaliana

RNA 5-methylcytosine (m5C) is implicated to have multiple biological and molecular roles in single cell and multicellular organisms, including regulation of mRNA translation and plant growth. However, there are still many questions waiting to be answered. In my research, I addressed the roles of m5C on rRNAs and tRNAs in Arabidopsis thaliana. In Chapter 2, NOP2, a putative rRNA m5C methyltransferase was shown to be genetically redundant and essential for ovule development. Three NOP2 orthologues, NOP2A, NOP2B and NOP2C were identified in the A. thaliana genome and are genetically redundant. I found nop2a nop2b mutants were lethal due to female gametophyte abortion at the two to eight-nucleate stages. Reduction of NOP2A in nop2b nop2c mutants using an artificial miRNA led to a range of post-embryo phenotypes, impaired ribosome activity and reduced m5C methylation of C2268 on the 25S rRNA. In Chapter 3, I tested tRNA cleavage in T2 RNase mutants, rns1, rns2, rns4 and wild type under oxidative stress by northern blotting. Small RNA sequencing suggested that loss of RNS2 results in changes of both pre-tRNA and mature tRNA profiles under oxidative stress. I also tested tRNA cleavage without m5C methyltransferase TRM4B, and without ribonuclease RNS2, and found m5C appears to protect tRNAAsp(GTC) from RNS2 cleavage. In Chapter 4, I explored the processing and molecular targets of tRNA halves originating from cleaved tRNAAsp(GTC). When a 35 nt 5’-half tRNAAsp fragment was expressed from a strong polymerase III promoter and the transgene was transformed into plants, no transgenic plants were recovered. Hence, we named this transgene, Killer. Subsequent transient expression of Killer in Nicotiana benthamiana leaves followed by small RNA sequencing and a targeted genetic suppressor screen in Arabidopsis indicated that the 35 nt 5’-half tRNAAsp in Killer was processed into several sRNAs, and involved RNA silencing proteins RDR6, DCL2, DCL3, DCL4, AGO1, AGO3, AGO4, AGO5, AGO7, AGO9 and AGO10. Two sRNAs from 5’-half tRNAAsp appear to silence embryo-defective genes, At1g67490 and At1g32490, thereby leading to embryo lethality. Taken together, my research addressed functions of m5C methyltransferases NOP2 and TRM4B, and revealed a role in the processing of tRNAAsp5’ in A. thaliana.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Analyse des PeBoW-Komplexes in der Ribosomenbiogenese

Während der Zellproliferation müssen Zellwachstum und Zellteilung koordiniert werden. Die Kopplung erfolgt in der Hefe durch einen Komplex aus Nop7p, Erb1p und Ytm1p, der sowohl an der Ribosomenbiogenese als auch an der Kontrolle der DNA-Replikation beteiligt ist. Die homologen Proteine Pes1, Bop1 und WDR12 werden in Säugern von Zielgenen des Transkriptionsfaktors c-Myc, einem zellulären Onkoprotein, kodiert. In dieser Arbeit wurde die Existenz eines evolutionär konservierten Komplexes aus Pes1, Bop1 und WDR12 (PeBoW-Komplex) in Säugern belegt. Dabei wurde gezeigt, dass Bop1 als zentrales Protein des Komplexes agiert und die Interaktion von Pes1 und WDR12 vermittelt. Die Integrität des Komplexes ist wesentlich für seine Funktion. Die Depletion einzelner Komponenten sowie die Überexpression des integrierenden Proteins Bop1 hemmen die Reifung der Vorläufer-rRNA der großen ribosomalen Untereinheit sowie die Proliferation der Zellen. Bop1-Überexpression führt zur Ausbildung von zwei Subkomplexen aus Bop1 und Pes1 bzw. Bop1 und WDR12. Während der Bop1/Pes1-Subkomplex als Teil der pre-Ribosomen im Nukleolus lokalisiert, wird WDR12 durch Bop1-Überexpression im Zytoplasma gehalten und fehlt im Nukleolus zur Ausbildung eines funktionellen PeBoW-Komplexes. Pes1 und WDR12 können unabhängig in den Nukleolus translozieren, während Bop1 dafür die Interaktion mit Pes1 benötigt. Untersuchungen zur Stabilität der einzelnen PeBoW-Komponenten zeigten, dass monomeres Bop1 extrem instabil ist, durch Inkorporation in den PeBoW-Komplex aber vor Abbau geschützt wird. Möglicherweise werden hierdurch interne PEST-Sequenzen in Bop1 maskiert. Die Menge an Bop1 ist somit abhängig von der Anwesenheit von Pes1 und WDR12. Die gegenseitige Abhängigkeit der Stabilität aller drei PeBoW-Komponenten konnte in weitergehenden Experimenten gezeigt werden. Schließlich wurde untersucht, ob der PeBoW-Komplex die Ribosomenbiogenese mit der DNA-Replikation über Interaktion mit dem ORC-Komplex, wie in der Hefe beschrieben, koordiniert. Mit Hilfe der BiFC-Methode konnte eine Interaktion von Pes1 mit Orc6, eines Faktors des ORC-Komplexes, gezeigt werden. Die koordinierende Funktion des PeBoW-Komplexes für Zellwachstum und Zellproliferation scheint von der Hefe bis zum Menschen stark konserviert zu sein