Uporaba alternativnega izrezovanja intronov za nadzor izražanja proteinov

Žvipelj, Mateja (author), Jerala, Roman (mentor), Dolinar, Marko (co-mentor)

Alternativno izrezovanje intronov in spajanje eksonov je aktualna raziskovalna tematika, o kateri je znanega že mnogo, pa vendar kljub številnim novim odkritjem še vedno obstaja precej neznank, ki na to razkritje še čakajo. Opisani so različni naravni mehanizmi tega procesa, mnogi pa so v raziskovalnih delih tudi inženirsko poustvarjeni. V tej magistrski nalogi smo, po zgledu v naravi prisotnih mehanizmov, želeli ustvariti sistem, s katerim bi usmerjali izrezovanje intronov in spajanje eksonov. To pomeni, da bi vplivali na končno obliko zrele mRNA, ki bi iz tako ustvarjenega konstrukta nastala. S tem bi ustvarili sistem za nadzorovano izražanje proteinov, kjer bi iz enega konstrukta omogočili nastanek več proteinov. V ta namen smo pripravili testni (osnovni) sistem treh eksonov in dveh intronov, ta konstrukt pa smo vstavili v plazmidno DNA in izražali v sesalskih celicah. Drugi in tretji ekson tega konstrukta sta vsebovala zapis za fluorescenčna proteina, prvi za protein mCIT, slednji za protein iRFP. V tako osnovanem sintetičnem genu sta omenjena proteina služila kot poročevalna proteina, njuno izražanje pa je bilo odvisno od načinov, na katere je proces izrezovanja in spajanja potekel. Ta osnovni konstrukt smo z uvajanjem novih elementov načrtovano spreminjali. Vpliv teh regulatornih elementov na proces izrezovanja in spajanja in s tem na izražanje obeh proteinov, smo spremljali z merjenjem fluorescence na pretočnem citometru ter z izolacijo in karakterizacijo molekul RNA in proteinov. Z eksperimentom smo želeli preveriti, ali vstavitev izbranih komplementarnih regij v obe intronski zaporedji privede do povečanega izražanja proteina iRFP in zmanjšanega nastanka proteina mCIT. Ugotovili smo, da se sistem odziva na dodane komplementarne regije. Opazno je namreč povišano izražanje proteina iRFP pri konstruktih z daljšimi komplementarnimi regijami, kar nakazuje na povečan delež nastale alternativne oblike mRNA, s katere se ta protein izraža. Obenem pa je videti, kot da se količina mRNA konstitutivne oblike, s katere se izraža mCIT, pri tem ne zmanjšuje kot bi pričakovali. Kljub temu lahko potrdimo, da z uvedbo komplementarnih zaporedij vplivamo na razmerje med dvema proteinoma, ki se izražata z zgoraj opisanega sistema, in da lahko razmerje do neke mere spreminjamo z dolžino tega komplementarnega zaporedja. V nadaljevanju bi bilo zanimivo preveriti, ali bi sedanja zasnova sistema delovala enako, če bi zapisa za fluorescenčna proteina nadomestili z zapisi za druge proteine. Z izgradnjo robustnejšega in zanesljivejšega sistema, ki bi natančno izražal želeno razmerje proteinov glede na vsebovano dolžino komplementarnega zaporedja, bi tak sistem lahko služil kot mehanizem za pripravo multiproteinskih kompleksov različnih proteinov iz enega konstrukta. Na tak način bi denimo lahko ustvarili celične receptorje ali virusne kapside, ki jih tvorijo proteini v različnih razmerjih. Raznovrstnost možnih aplikacij se kaže tudi v tem, da bi tak sistem, z odzivanjem na spremembo dejavnikov v okolici, kjer bi se nahajal, pridobil zanimive terapevtske razsežnosti.

• Language: Slovenian
• Keywords: izrezovanje intronov in spajanje eksonov, alternativno izrezovanje, nadzorovano izražanje proteinov, komplementarna zaporedja
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-144529
• COBISS.SI-ID: 143944707
• Publication date in RUL: 28.02.2023

Secondary language

• Language: English
• Title: Use of alternative splicing of introns for protein expression control

Abstract

Alternative splicing is a current scientific topic that is well understood, however even with many of new discoveries, there is still much to be revealed. Numerous mechanisms of this process are well described and some of them were also recreated in an engineering way. By mimicking the naturally existing mechanisms, in the present master’s thesis we aimed to set up a protein expression control system by which we would be able to influence the expression of proteins from a single construct. That is, it would be possible to control the types of mature mRNAs formed from the same pre-mRNA molecules. We created a test system consisting of three exons and two introns, this construct was subsequently inserted into plasmid DNA and expressed in mammalian cells. In the middle of the second exon the mCIT coding sequence was added, and the third exon contained the sequence encoding the iRFP protein, respectively. By changing this system with newly incorporated elements, we analysed their effects on the splicing process by fluorescence measurements using a flow cytometer. Analyses were also performed at the RNA and protein levels. Our goal was to investigate whether the insertion of homologous sequences in both intron regions of a construct promotes iRFP expression and simultaneously influences on the lower expression of mCIT. We confirmed the effect of homologous regions on the splicing process of the system described. Indeed, the iRFP fluorescence of constructs with longer homologous sequences increased, suggesting that a higher amount of alternative mRNA was formed from pre-mRNA molecules. At the same time the drop in mCIT expression was not detected, consistent with results observed at the mRNA level. Nevertheless, by incorporating homologous sequences, we confirmed that the ratio of two proteins expressed from the same transcript can be controlled. Moreover, we showed that the effect can be regulated with the length of the homologous region in both introns. In the light of future perspectives, it would be interesting to test whether the created system also works well when the sequences of the fluorescent proteins are substituted with any other protein sequences. The presented system offers a variety of interesting applications, especially in the aspects where the proportion of protein components plays a crucial role. A more robust and reliable system with the ability to fine-tune the ratio of different proteins would allow us to use it for the production of multiprotein complexes expressed from the same construct. For example, the system has a potential to be used for recombinant cell receptors or viral capsid production. By incorporating the environmental sensitivity of the system, it would be possible to extend it for various therapeutic applications.

• Keywords: splicing, alternative splicing, protein expression control, homologous sequences