prikaz prve stranice dokumenta Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure
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master's thesis
Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure
2023. urn:nbn:hr:193:703125
Jurković, Borna
University of Rijeka
Faculty of Biotechnology and Drug Development

Cite this document

Jurković, B. (2023). Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure (Master's thesis). Rijeka: University of Rijeka. Retrieved from https://urn.nsk.hr/urn:nbn:hr:193:703125

Jurković, Borna. "Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure." Master's thesis, University of Rijeka, 2023. https://urn.nsk.hr/urn:nbn:hr:193:703125

Jurković, Borna. "Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure." Master's thesis, University of Rijeka, 2023. https://urn.nsk.hr/urn:nbn:hr:193:703125

Jurković, B. (2023). 'Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure', Master's thesis, University of Rijeka, accessed 26 April 2024, https://urn.nsk.hr/urn:nbn:hr:193:703125

Jurković B. Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure [Master's thesis]. Rijeka: University of Rijeka; 2023 [cited 2024 April 26] Available at: https://urn.nsk.hr/urn:nbn:hr:193:703125

B. Jurković, "Effect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure", Master's thesis, University of Rijeka, Rijeka, 2023. Available at: https://urn.nsk.hr/urn:nbn:hr:193:703125

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Metadata
TitleEffect of disrupting the mRNA turnover on the genome-wide RNA 3'-end structure
Title (croatian)Utjecaj poremećaja u raspadu mRNA na strukturu RNA 3 kraja na razini genoma
AuthorBorna Jurković
MentorMichal Malecki (mentor)
MentorChristian Andrew Reynolds (komentor)
Committee memberMichal Malecki (predsjednik povjerenstva)
Committee memberChristian Andrew Reynolds (član povjerenstva)
Committee memberRozi Andretić Waldowski (član povjerenstva)
Committee memberNela Malatesti (član povjerenstva)
Committee memberKatarina Kapuralin (član povjerenstva)
GranterUniversity of Rijeka
(Faculty of Biotechnology and Drug Development)
Rijeka
Defense date and country2023-07-14, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
Uridylation is a common widespread 3'-end modification in eukaryotes; however, its impact on mRNA fate is not clear. The mRNA decay mechanism in S. pombe is quite similar to that in humans, where the first step usually involves uridylation of the 3´-end of mRNA by terminal uridyltransferases (TUTases). The aim of this project was to investigate the effects on mRNA decay and mRNA tails when gene expression levels of the factors involved in mRNA decay are manipulated. We decided to delete the enhancer of mRNA decapping gene, edc1, and the exonuclease gene xrn1, while overexpressing the terminal uridyltransferases genes, cid1 and cid16. The transformations were based on the cellular intake of a linear cassette and homologous recombination of the same cassette. The transformed strains were verified using colony-PCR, and overexpression strains were additionally verified using western blotting. Subsequently, mRNA was isolated and purified, and NGS libraries were prepared using a custom GW3´RACE (TAIL-seq) protocol. The samples were then sent for paired-end Illumina sequencing. The sequencing data was analyzed using bioinformatic tools, and included read trimming, filtering and genome alignment. Although the libraries for strains overexpressing cid1 and cid16 did not work, the cid16 expression dependent strain showed a growth defect. Other main findings were as follows: deletion of edc1 or xrn1 resulted in the shortening of mRNA tail length, increased mRNA uridylation, and accumulation of non-tailed mRNAs. In other words, the deletion of edc1 or xrn1 produced the same results, namely the accumulation of mRNA decay intermediaries. Additionally, the differential gene expression analysis revealed six commonly up-regulated genes between the mutants, with the most up-regulated gene being ubi4, which encodes for ubiquitin.
Abstract (croatian)
Uridilacija je česta modifikacija 3'-kraja u eukariotima; međutim, njen utjecaj na sudbinu mRNA nije jasan. Mehanizam raspada mRNA kod S. pombe prilično je sličan onom kod ljudi, gdje prvi korak obično uključuje uridilaciju 3´-kraja mRNA terminalnim uridiltranferazama (TUTazama). Cilj ovog projekta bio je istražiti učinke na raspad mRNA i repove mRNA kada se manipuliraju ekspresije gena faktora uključenih u raspad mRNA. Odlučili smo izbrisati gen edc1, koji je pojačavač dekapiranja mRNA, i gen xrn1, koji kodira egzonukleazu, dok smo prekomjerno izražavali gene terminalnih uridiltranferaza, cid1 i cid16. Transformacije su se temeljile na staničnom unosu linearnog kasetnog elementa i homologne rekombinacije iste kasete. Transformirani sojevi su potvrđeni korištenjem colony-PCR-a, a sojevi s prekomjernim izražavanjem su dodatno potvrđeni korištenjem western blot analize. Nakon toga je mRNA izolirana i pročišćena, a NGS libraries su pripremljene korištenjem prilagođenog GW-3´RACE (TAIL-seq) protokola. Uzorci su zatim poslani na paired-end sekvenciranje Illumina tehnologijom. Podaci sekvenciranja su analizirani korištenjem bioinformatičkih alata, uključujući podrezivanje očitanja, filtriranje i poravnanje s referentnim genomom. Iako biblioteke za sojeve s prekomjernim izražavanjem cid1 i cid16 nisu funkcionirale, soj s prekomjernim izražavanjem cid16 je pokazao defekt rasta. Ostali glavni rezultati su sljedeći: brisanje edc1 ili xrn1 rezultiralo je skraćivanjem duljine repova mRNA, povećanom uridilacijom mRNA i nakupljanjem mRNA bez repova. Drugim riječima, brisanje edc1 ili xrn1 proizvelo je iste rezultate, odnosno nakupljanje intermediarnih produkata mRNA raspada. Također, analiza diferencijalnog izraza gena je otkrila šest zajednički up-reguliranih gena između mutanata, pri čemu je najviše up-regulirani gen bio ubi4, koji kodira ubiquitin.
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:193:703125
Study programmeTitle: Biotechnology in medicine
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra biotehnologije u medicini (magistar/magistra biotehnologije u medicini)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2023-10-11 12:43:04