Title Effect 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 Author Borna Jurković Mentor Michal Malecki (mentor) Mentor Christian Andrew Reynolds (komentor)Committee member Michal Malecki (predsjednik povjerenstva) Committee member Christian Andrew Reynolds (član povjerenstva)Committee member Rozi Andretić Waldowski (član povjerenstva)Committee member Nela Malatesti (član povjerenstva)Committee member Katarina Kapuralin (član povjerenstva)Granter University of Rijeka Defense date and country 2023-07-14, Croatia Scientific / art field, BIOTECHNICAL SCIENCES 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
mRNA decay
urydilation
S. pombe
Keywords (croatian)
raspad mRNA
uridilacija
S. pombe
Language english URN:NBN urn:nbn:hr:193:703125 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2023-10-11 12:43:04
