Structural studies of CGAG-rich sequences from regulatory regions of genes implicated in neurodevelopment

Novotny, Aleš (avtor), Plavec, Janez (mentor)

The AUTS2 gene has been shown to influence neurodevelopment by controlling the number of neurons, influencing neuronal migration and promoting axon and dendrite growth. The AUTS2 protein can be expressed either as full-length or C-terminal isoform. Precise regulation of expression of both isoforms is crucial for brain development. The molecular mechanism responsible for the expression regulation of the two isoforms is unknown and no mechanism has been proposed. In this work, we have identified a CGAG-rich region in the promoter of AUTS2 gene. The CGAG-rich region spans 60 nucleotides and contains a putative protein binding site (PPBS), d(AGCGAAAGCACGAA). Using NMR, UV-Vis spectroscopy and MD simulations, we show that oligonucleotides derived from this region adopt thermally stable non-canonical hairpin structures. We uncover a mechanism, which we refer to as a four-nucleotide register shift, that enables the formation of a variety of hairpins depending on the number of CGAG repeats in the oligonucleotide sequence. The hairpins are stabilized by G:C and sheared G:A base pairs that are arranged in structural motifs that we term CGAG blocks. We observe significant structural differences between the loop regions of these non-canonical hairpin structures. We show that epigenetic modifications 5-methylcytosine and 5-hydroxymethylcytosine incorporated in CGAG-rich oligonucleotide result in marginal increase and minor decrease in thermal stability, respectively. The modifications cause only local structural changes. We also examine an oligonucleotide that is complementary to the CGAG-rich strand and show that the oligonucleotide d(CGCT)4 adopts a thermally rather labile hairpin structure stabilized by G:C and C:T base pairs. We conclude that a transient formation of non-canonical hairpin structures by CGAG-rich oligonucleotides is possible in vivo. Therefore, our work provides an insight into a plausible mechanism responsible for regulation of expression of the two AUTS2 isoforms.

• Jezik: Angleški jezik
• Ključne besede: -
• Vrsta gradiva: Doktorsko delo/naloga
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-154834
• COBISS.SI-ID: 188160259
• Datum objave v RUL: 05.03.2024

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Strukturne študije s CGAG bogatih zaporedij iz regulatornih regij genov, vpletenih v nevrološki razvoj

Izvleček

Gen AUTS2 vpliva na razvoj živčevja, saj uravnava število nevronov, vpliva na migracijo nevronov ter spodbuja rast aksonov in dendritov. Protein AUTS2 se lahko izraža v izoobliki polne dolžine ali pa v izoobliki, ki ustreza C-končnemu delu proteina. Natančno uravnavanje izražanja obeh izooblik je ključnega pomena za razvoj možganov. Molekularni mehanizem, ki je odgovoren za uravnavanje izražanja obeh izooblik, še ni znan, prav tako pa zaenkrat še ni bila predlagana nobena hipoteza v zvezi s tem mehanizmom. Tekom doktorata smo v promotorju gena AUTS2 identificirali regijo, bogato s CGAG. Regija, bogata s CGAG, obsega 60 nukleotidov in vsebuje domnevno vezavno mesto za proteine (PPBS), d(AGCGAAAGCACGAA). Z uporabo NMR, UV-Vis spektroskopije in MD simulacij smo pokazali, da imajo oligonukleotidi, pridobljeni iz te regije, termično stabilne, nekanonične lasnične strukture. Razkrili smo mehanizem štiri-nukleotidnega zamika verige, ki odvisno od števila ponovitev CGAG v nukleotidnem zaporedju oligonukleotida, omogoča nastanek različnih lasnic. Lasnice stabilizirajo bazni pari G:C in G:A, ki so razporejeni v strukturne motive imenovane bloki CGAG. V omenjenih nekanoničnih lasnicah smo opazili pomembne strukturne razlike med območji zank. Pokazali smo, da epigenetski modifikaciji 5-metilcitozin in 5-hidroksimetilcitozin, vključeni v oligonukleotid, bogat s CGAG, povzročita minimalno povišanje in majhno znižanje termične stabilnosti. Modifikaciji povzročita le lokalne strukturne spremembe. Proučili smo tudi komplementarno verigo CGAG bogate regije in pokazali, da oligonukleotid d(CGCT)4 tvori termično precej labilno lasnico, ki jo stabilizirajo bazni pari G:C in C:T. Sklepamo, da bi oligonukleotidi, bogati s CGAG, lahko prehodno tvorili nekanonične lasnične strukture in vivo. Naše delo zato omogoča vpogled v verjeten mehanizem, ki je odgovoren za uravnavanje izražanja obeh izooblik AUTS2.

• Ključne besede: -