Določanje proteinov v okolici STAU1 in STAU2 v celicah HEK293

Lenarčič, Katarina (author), Petrovič, Uroš (mentor), Milek, Miha (comentor)

Protein staufen je RNA-vezavni protein, ki skupaj z molekulo RNA tvori ribonukleoproteinske komplekse kot so na primer stresne granule, ki omogočajo preživetje stresnih pogojev. Spremembe RNA-vezavnih proteinov lahko povzročijo številne nevrodegenerativne bolezni, za katere je značilno propadanje nevronov. V naši raziskavi smo z metodo označevanja okolice z biotinom označili proteine, ki interagirajo ali so v bližini proteina staufen. Uporabili smo dve biotin-ligazi: BirA* in TurboID. Ko smo dokazali, da se je fuzijski protein sintetiziral v celicah HEK293 in je bila biotinilacija proteinov uspešna smo izolirane proteine poslali na analizo z masno spektrometrijo. Rezultati so pokazali, da večina proteinov, ki smo jih določili, sodeluje pri vezavi RNA ali pri strukturi ribosoma, kar nekaj proteinov pa se veže z molekulo DNA. Večje razlike med proteini v okolici proteinov staufen 1 in staufen 2 nismo zaznali. Uporaba različnih biotin-ligaz pa nam je dala vpogled v časovno dinamiko interakcij. Pokazali smo, da se protein staufen nahaja v okolici ribosoma in molekule RNA.

• Language: Slovenian
• Keywords: nevrodegenerativne bolezni, protein staufen, interaktor, biotin-ligaza
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publisher: [K. Lenarčič]
• Year: 2023
• PID: 20.500.12556/RUL-145087
• UDC: 577.2
• COBISS.SI-ID: 149656067
• Publication date in RUL: 05.04.2023

LENARČIČ, Katarina, 2023, Določanje proteinov v okolici STAU1 in STAU2 v celicah HEK293 [online]. Master’s thesis. K. Lenarčič. [Accessed 21 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=145087

Secondary language

• Language: English
• Title: Identification of proteins in the proximity of STAU1 and STAU2 in HEK293 cells

Abstract

Staufen protein is an RNA-binding protein that forms ribonucleoprotein complexes with the RNA, e.g., stress granules which help survive stress conditions. Changes of RNA-binding proteins can cause a number of neurodegenerative diseases characterised by neuronal degeneration. In our study, we used a proximity labelling method to label proteins that interact or are in the vicinity of the Staufen protein. Two biotin ligases were used: BirA* and TurboID. After confirming that the fusion protein was correctly expressed and that the biotinylation of the proteins was successful, we sent the isolated proteins for analysis by mass spectrometry. We concluded that most of the proteins we identified are involved in RNA binding or ribosome structure, and few proteins bind to DNA. We did not detect any major difference between the proteins surrounding Staufen 1 and Staufen 2 proteins. Using different biotin-ligases gave us insight into the temporal dynamics of the interactions. We showed that the Staufen protein is located near the the ribosome and the RNA molecule.

• Keywords: neurodegenerative diseases, staufen protein, interactor, biotin ligase