Uloga nekodirajuće transkripcije u regulaciji ekspresije gena PHO5 kvasca Saccharomyces cerevisiae

undergraduate thesis

• Title (english): Role of non coding transcription in regulation of yeast Saccharomyces cerevisiae PHO5 gene expression
• Author: Jurica Ljubas
• Mentor: Igor Stuparević (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2021-07-15, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Food Technology

Abstract

Antisense RNA su nekodirajuće RNA koje nastaju inicijacijom transkripcije s 3' kraja kodirajućeg lanca gena. Ove su molekule kratkog vremena poluživota, budući da su ubrzo nakon početka transkripcije terminirane NNS kompleksom i degradirane RNA egzosomom. Brojna istraživanja pokazuju da se antisense RNA transkribiraju na velikom broju lokusa i da se njihova uloga povezuje s regulacijom ekspresije pripadajućih gena. Cilj ovog rada bio je istražiti ulogu antisense transkripcije u regulaciji ekspresije gena PHO5 kvasca S. cerevisiae. Sustavom Anchor away postignut je rapamicinom-inducirani eksport RNA-vezujuće podjedinice NNS kompleksa Nrd1 iz jezgre, što je rezultiralo sporijom kinetikom aktivacije transkripcije gena PHO5. Nadalje, korišten je CRISPRi sustav sačinjen od katalički-inaktivne endonukleaze Cas9 (dCas9) i molekule guide RNA, koja navodi dCas9 na specifičnu regiju gena. CRISPRi sustavom ciljano je zaustavljena transkripcija PHO5 antisense RNA, što je rezultiralo bržom kinetikom aktivacije transkripcije gena PHO5. Dobiveni rezultati stoga upućuju na negativan regulatorni utjecaj antisense RNA na kinetiku ekspresije gena PHO5.

Abstract (english)

Antisense RNAs are defined as non-coding RNAs which are made upon transcriptional initiation from the 3' ends of genes. These molecules have short half-lives considering that, soon after the start of their transcription, they are terminated by the NNS complex and degraded by the RNA exosome. However, numerous studies have shown that antisense RNAs are transcribed at a large number of loci and that they could play important roles in transcriptional regulation of corresponding genes. The aim of this work was to explore the role of antisense transcription in regulation of yeast S. cerevisiae PHO5 gene expression. Using the Anchor away system, we achieved rapamycin-induced export of the NNS complex RNA-binding subunit Nrd1, which resulted in slower kinetics of PHO5 gene transcriptional activation. Furthermore, we used CRISPRi system comprised of the catalitically-inactive endonuclease Cas9 (dCas9) and guide RNA, which guides dCas9 protein to a specific gene region. Using CRISPRi, we specifically blocked only antisense transcription at the PHO5 gene locus, which resulted in more rapid kinetics of PHO5 gene transcriptional activation. These results indicate that antisense transcription has a negative effect on PHO5 gene expression kinetics.

• Keywords: antisense RNA; regulacija ekspresije gena; gen PHO5; NNS kompleks; RNA egzosom
• Keywords (english): antisense RNAs; transcriptional regulation; PHO5 gene; NNS complex; RNA exosome
• Language: croatian
• URN:NBN: urn:nbn:hr:159:177002
• Study programme: Title: Food Technology; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka prehrambene tehnologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) inženjer/inženjerka prehrambene tehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Repository: Repository of the Faculty of Food Technology and Biotechnology
• Created on: 2021-07-19 10:20:46