Molekulsko kloniranje in filogenetske analize proteina matrin 3

Osolin, Manca (author), Župunski, Vera (mentor)

Matrin 3 (MATR3) je DNA- in RNA-vezavni protein jedrnega matriksa. Sodeluje pri uravnavanju transkripcije, alternativnem izrezovanju intronov, izvozu mRNA iz jedra in stabilizaciji mRNA. Mutacije MATR3 vplivajo na razvoj nevrodegenerativnih bolezni ALS (amiotrofična lateralna skleroza) in FTD (frontotemporalna demenca). Za te bolezni je značilna povečana koncentracija MATR3 v jedru, nepravilna lokalizacija v citoplazmi ali pa tvorba agregatov v citoplazmi. MATR3 z mutacijo S85C inhibira nastanek stresnih granul, ki se tvorijo kot fiziološki odziv na celični stres, in tako prispeva k patologiji ALS. Diplomsko delo je sestavljeno iz laboratorijskega in bioinformatskega dela. V okviru laboratorijskega dela smo z metodo kloniranja IVA izvedli delecijo zapisa za BioID2 iz vektorjev pcDNA3.1-MATR3-BioID2-HA in pcDNA3.1-ALS-MATR3-BioID2-HA ter tako pridobili vektorja pcDNA3.1-MATR3-HA in pcDNA3.1-ALS-MATR3-HA. Te vektorje bi lahko v nadaljevanju uporabili za (ko)transfekcijo sesalskih celičnih linij in na ta način preverili, kako izražanje MATR3 in MATR3 z mutacijo S85C, ki je značilna za ALS, vpliva na nastanek stresnih granul. V okviru bioinformatskega dela pa smo analizirali filogenijo MATR3. Preučili smo evolucijo MATR3 znotraj sesalcev in jo primerjali z evolucijo sesalskih vrst. Ugotovili smo, da se filogenetski odnosi MATR3 v glavnem ujemajo z evolucijo sesalskih vrst. Najboljše ujemanje z evolucijo sesalcev smo dobili pri filogenetskem drevesu na podlagi nukleotidnih zaporedij mRNA MATR3. Pri filogenetski analizi na podlagi aminokislinskih zaporedij MATR3 pri sesalcih so bile namreč vrednosti pristopa ponovnega vzorčenja (angl. bootstrap) prenizke, da bi bili evolucijski odnosi zanesljivi. Razvrstitev določenih redov v nasprotju s privzeto evolucijo sesalcev smo skušali razložiti tako, da smo poravnali izbrana nukleotidna zaporedja in identificirali mesta odstopanj. Analizirali smo tudi evolucijo MATR3 znotraj vretenčarjev, kjer smo najboljše filogenetsko drevo zaradi bolj raznolikih zaporedij dobili z uporabo aminokislinskih zaporedij.

• Language: Slovenian
• Keywords: matrin 3, ALS, stresne granule, molekulsko kloniranje, filogenetska analiza
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-130005
• COBISS.SI-ID: 82770947
• Publication date in RUL: 10.09.2021

Secondary language

• Language: English
• Title: Molecular cloning and phylogenetic analyses of matrin 3

Abstract

Matrin 3 (MATR3) is a DNA- and RNA-binding nuclear matrix protein. It contributes to the regulation of transcription, alternative intron splicing, mRNA export from the nucleus and mRNA stabilization. Mutations in MATR3 have been linked to the development of some neurodegenerative diseases such as ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia). These diseases are characterized by increased nuclear concentration of MATR3, its irregular localization in the cytoplasm or aggregate formation. This thesis consists of laboratory work and bioinformatic work. Within the scope of the laboratory work, we deleted the BioID2 marker from vectors pcDNA3.1-MATR3-BioID2-HA and pcDNA3.1-ALS-MATR3-BioID2-HA using the IVA cloning method and thus produced vectors pcDNA3.1-MATR3-HA and pcDNA3.1-ALS-MATR3-HA. MATR3 with mutation S85C inhibits the formation of stress granules, which form as a result of a physiological response to cellular stress, and thus contributes to ALS pathology. We could have further used these vectors for (co)transfection of mamallian cell lines. In this way, we could have determined how MATR3 and MATR3 with mutation S85C, which is characteristic of ALS, affect the formation of stress granules. Within the scope of bioinformatic work, we analyzed the phylogeny of MATR3. We examined the evolution of MATR3 in mammals and compared it to the evolution of mammalian species. We found that the phylogenetic relationships of MATR3 mostly match the evolution of mammalian species. The phylogenetic tree based on the nucleotide sequences of MATR3 mRNA showed the best matching with mammalian evolution. In the phylogenetic analysis based on the amino acid sequences of MATR3 in mammals, the bootstrap values were too low to reliably determine the evolutionary relationships. We tried to explain the phylogenetic relationships of specific orders that were inconsistent with mammalian evolution by aligning these nucleotide sequences and identifying divergence points. We also analyzed the evolution of MATR3 in vertebrates, where the best phylogenetic tree was obtained using amino acid sequences, as these sequences were more diverse.

• Keywords: Matrin 3, ALS, stress granules, molecular cloning, phylogenetic analysis