Analiza stabilnosti mRNA i određivanje mRNA-veznih proteina kod nedostatne aktivnosti S-adenozilhomocistein-hidrolaze

master's thesis

• Title (english): Identification of mRNA binding proteins and determination of mRNA stability in AHCY deficiency
• Author: Filip Rokić
• Mentor: Jasmina Rokov Plavec (mentor)
• Mentor: Oliver Vugrek (mentor)
• Committee member: Jasmina Rokov Plavec (predsjednik povjerenstva)
• Committee member: Nives Galić (član povjerenstva)
• Committee member: Josip Požar (član povjerenstva)
• Committee member: Marko Močibob (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2016-02-29, 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: degradacija mRNA; mutirana mRNA; protein SAHH; mutageneza
• Keywords (english): AHCY; mutant mRNA; mRNA decay; mutagenesis
• Language: croatian
• URN:NBN: urn:nbn:hr:217:401540
• Study programme: Title: 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 resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2018-03-01
• Created on: 2017-03-14 16:49:20