prikaz prve stranice dokumenta Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze
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master's thesis
Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze
Zagreb: University of Zagreb, Faculty of Science, 2016. urn:nbn:hr:217:401540
Rokić, Filip
University of Zagreb
Faculty of Science
Department of Chemistry

Institutional repository: Repository of the Faculty of Science

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Rokić, F. (2016). Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:401540

Rokić, Filip. "Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze." Master's thesis, University of Zagreb, Faculty of Science, 2016. https://urn.nsk.hr/urn:nbn:hr:217:401540

Rokić, Filip. "Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze." Master's thesis, University of Zagreb, Faculty of Science, 2016. https://urn.nsk.hr/urn:nbn:hr:217:401540

Rokić, F. (2016). 'Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze', Master's thesis, University of Zagreb, Faculty of Science, accessed 23 December 2024, https://urn.nsk.hr/urn:nbn:hr:217:401540

Rokić F. Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2016 [cited 2024 December 23] Available at: https://urn.nsk.hr/urn:nbn:hr:217:401540

F. Rokić, "Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2016. Available at: https://urn.nsk.hr/urn:nbn:hr:217:401540

Metadata
TitleAnaliza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze
Title (english)Identification of mRNA binding proteins and determination of mRNA stability in AHCY deficiency
AuthorFilip Rokić
MentorJasmina Rokov Plavec (mentor)
MentorOliver Vugrek (mentor)
Committee memberJasmina Rokov Plavec (predsjednik povjerenstva)
Committee memberNives Galić (član povjerenstva)
Committee memberJosip Požar (član povjerenstva)
Committee memberMarko Močibob (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Chemistry)
Zagreb
Defense date and country2016-02-29, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Chemistry
Abstract
Nedostatna aktivnost S-adenozilhomocistein-hidrolaze (SAHH) je nasljedno metaboliĉko oboljenje uzrokovano mutacijom u genu koja dovodi do inaktivacije proteina SAHH te poremećaja staniĉnih metilacijskih procesa. Nedavno je u ovom genu otkrivena nova mutacija koja kodira za verziju proteina SAHH_Y328D i koja uzrokuje gotovo potpuni nedostatak molekule mRNA te smanjenu ekspresiju proteina iz dosad nepoznatog razloga. Navedena mutacija ne uvodi preuranjeni STOP-kodon niti dokida postojeći, stoga su staniĉni mehanizmi koji uklanjaju takve transkripte iskljuĉeni. Hipoteza ovog rada ukljuĉuje moguće promjene u sekundarnoj strukturi mutiranog transkripta uzrokovane toĉkastom mutacijom koje dovode do aktivacije manje istraženog mehanizma degradacije uslijed zastajanja ribosoma tijekom translacije (eng. no-go decay, NGD). U cilju utvrđivanja mehanizma degradacije, in vitro mutagenezom uvedena je mutacija u molekulu cDNA koja kodira za protein SAHH, te je korištenjem lanĉane reakcije polimerazom (PCR) dobiven kalup za in vitro transkripciju. Instrumentnim metodama cirkularnog dikroizma (CD) i diferencijalno skenirajućom kalorimetrijom (DSC) utvrđene su razlike u sekundarnim strukturama i temperaturnoj stabilnosti sintetiziranih molekula mRNA. Molekule mRNA divljeg tipa odnosno verzije mRNA koja kodira za protein SAHH_Y328D, obilježene su detiobiotinom te su iskorištenje za izdvajanje RNA-vezujućih proteina iz staniĉnog lizata HepG2 stanica. Izolirani proteini identificirani su spektrometrijom masa (MS) a iz diferencijalnih proteinskih profila predložen je najvjerojatniji mehanizam razgradnje molekule mRNA kod pacijenta s navedenom mutacijom.
Abstract (english)
S-adenosylhomocysteine hydrolase (AHCY) deficiency is an inherited metabolic disorder caused by a mutation of the AHCY gene, which significantly reduces enzymatic activity and alters the cellular methylation potential. Recently, a novel mutation (p.Y328D) was discovered that causes its transcript to degrade leading to highly reduced expression of AHCY protein. The single nucleotide polymorphism (SNP) does not introduce a premature STOP codon nor does it affect the actual STOP codon so the cellular mechanisms that degrade such transcripts can be excluded. Main hypothesis of this paper includes changes in the secondary structure of the transcript caused by the SNP. Those changes of the overall structure could cause stalling of ribosomes during translation of the mutant transcript and activate a no-go decay (NGD) mechanism. Identical SNP was introduced into cDNA sequence of AHCY using site-directed mutagenesis. Templates for in vitro transcription were synthesized using polymerase chain reaction (PCR). Circular dichroism (CD) and differential scanning calorimetry (DSC) measurements were performed to show that the SNP introduces changes to the secondary structure and influences thermal stability of the mutant transcript. mRNAs were labeled on their 3' end with desthiobiotin and used for RNA-protein pull-down assay using HepG2 cell lysates. After enrichment of the RNA-binding proteins (RBPs) samples were analyzed using mass spectrometry (MS). On the basis of the identified proteins a most probable degradation mechanism was proposed for the mutant AHCY mRNA.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:401540
Study programmeTitle: Graduate university studies in chemistry; specializations in: Research programme
Course: Research programme
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra kemije (magistar/magistra kemije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Embargo expiration date: 2018-03-01
Terms of useLicence In copyright icon
Created on2017-03-14 16:49:20