Utjecaj narušavanja gena HBS1L, PELO, NONO, SND1 na mRNA koja kodira mutirani protein S-adenozilhomocistein-hidrolazu

master's thesis

• Title (english): The effect of HBS1L, PELO, NONO and SND1 knock-out on mutated S-adenosylhomocysteine hydrolase mRNA
• Author: Ena Šimunić
• Mentor: Oliver Vugrek (mentor)
• Committee member: Jasmina Rokov Plavec (predsjednik povjerenstva)
• Committee member: Ivan Kodrin (član povjerenstva)
• Committee member: Snežana Miljanić (član povjerenstva)
• Committee member: Ivica Đilović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2020-02-17, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

S-adenozilhomocistein-hidrolaza (SAHH) je enzim koji katalizira reakciju hidrolize Sadenozil-
L-homocisteina na adenozin i homocistein. SAHH ima ključnu ulogu u održavanju
metilacijskog potencijala u stanici te njegov nedostatak dovodi do teškog metaboličkog
poremećaja. Preliminarni podaci pokazuju da jedna od dosad opisanih mutacija (c.982 T>G),
uz smanjenje aktivnosti enzima uzrokuje i smanjenje razine SAHH mRNA uz smanjenu
razinu proteina SAHH u stanicama pacijenta. Pretpostavlja se da je mehanizam razgradnje
uslijed zastajanja ribosoma tijekom translacije vjerojatni mehanizam razgradnje te mRNA. U
svrhu potvrde hipoteze generirane su četiri stanične linije u kojima su metodom CRISPRCas9
pojedinačno narušeni geni HBS1L, PELO, NONO i SND1, potencijalni ključni čimbenici
u razgradnji mRNA koja kodira mutirani protein SAHH, te je analiziran utjecaj njihovog
narušavanja na razinu ekspresije te mRNA. Rezultati analize razine SAHH mRNA u
staničnim linijama s narušenim genima metodom RT-qPCR pokazuju da jedino u staničnoj
liniji s narušenim genom NONO dolazi do povrata razine ekspresije mutiranog transkripta.

Abstract (english)

S-adenosylhomocystein hydrolase (AHCY) is an enzyme that catalyses hydrolysis of Sadenosyl-
L-homocysteine to adenosine and homocysteine. AHCY has the key role in
maintaining cells' methylation potential, and its deficiency leads to severe metabolic disorder.
Preliminary data shows that one of the mutations described so far (c.982 T>G) leads to
decrease of the enzyme function and also causes a decrease of AHCY mRNA levels together
with decreased AHCY protein levels in patient cells. It can be assumed that no-go decay is the
probable mechanism of AHCY mRNA decay. To test this hypothesis, four knock-out cell
lines have been generated. Using a CRISPR-Cas9 mediated approach, four genes, e.g.
HBS1L, PELO, NONO and SND1 have been proposed as potential key factors in AHCY
mRNA decay. Subsequently, all aforementioned genes have been individually knocked-out,
and knock-out effects on AHCY mRNA expression levels have been analysed. RT-qPCR
results show an expected rise in AHCY mRNA levels only in NONO knock-out cell line.

• Keywords: CRISPR-Cas9; degradacija mRNA; HBS1L; NONO; PELO; SAHH; SND1
• Keywords (english): AHCY deficiency; CRISPR-Cas9; mRNA degradation; HBS1L; NONO; PELO; SND1.
• Language: croatian
• URN:NBN: urn:nbn:hr:217:244028
• 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: 2020-08-17
• Created on: 2020-02-21 12:12:59