Title Rare codon differences affect expression of the neurodevelopmental protein paralogs NDE1 and NDEL1
Title (croatian) Razlike u rijetkim kodonima utječu na ekspresiju neurorazvojnih proteinskih paraloga NDE1 i NDEL1
Author Matea Kršanac
Mentor Nicholas Bradshaw (mentor)
Committee member Nicholas Bradshaw (član povjerenstva)
Committee member Mladen Merćep (član povjerenstva)
Committee member Jelena Ban (predsjednik povjerenstva)
Granter University of Rijeka (Faculty of Biotechnology and Drug Development) Rijeka
Defense date and country 2023-09-27, Croatia
Scientific / art field, discipline and subdiscipline BIOTECHNICAL SCIENCES Biotechnology
Abstract Chronic mental illnesses are persistent and disrupt cognition, emotion regulation and behaviour, and their exact causes and mechanisms remain largely unknown. Previous studies identified several genetic factors that play a role in their development. Nuclear Distribution Element 1 (NDE1) and Nuclear Distribution Element-Like 1 (NDEL1) are proteins that arise form a gene duplication event and are vital in cell mitosis and neurodevelopment. They have been associated with brain malformations and neurodevelopmental disorders. Although being structurally similar, they exhibit distinct pathophysiological functions.
Rare codon bias is a tendency for some codons to be more frequently used than others in a specific species, while they encode for the same amino acid. Codon rarity has been demonstrated to affect translation speed, protein folding, translational control, and protein expression levels in multiple known genomes. In human NDE1 and NDEL1 genes, the latter shows an increase in frequency of rare codon usage.
In this thesis, I first investigated whether the difference in codon rarity between NDE1 and NDEL1 was conserved across multiple species that represented major vertebrate genera. Next, I explored if altering the codons responsible for encoding NDE1 and NDEL1 could mitigate the differences in expression levels between these two proteins. To achieve this, I examined NDE1 constructs that used codons closely, matching ones found in NDEL1, while maintaining the correct amino acid sequence, and vice versa. Finally, I examined the expression patterns of both wild type and switched codon NDE1 and NDEL1 proteins by fluorescent microscopy with the aim of identifying any noticeable distinctions if such differences existed.
The preference of NDEL1 for rare codon usage that had previously been seen in humans, was also seen in non-human primates and non-primate mammals, suggesting the rare codon bias differences between the two genes to be conserved across mammalian species. Our findings indicated that the difference in codon rarity in wild type NDE1 and NDEL1 had an impact on their respective expression levels, with NDE1 exhibiting higher expression within cells. Notably, when examining constructs with switched codons, there was indication of this difference being nullified. This observation suggests that the use of more commonly occurring codons could potentially reverse the trend of lower protein expression levels and that rare codon bias may be partially responsible for the differing functions of the two proteins.
Abstract (croatian) Kronične mentalne bolesti su dugotrajne i ometaju kogniciju, regulaciju emocija i ponašanje, a njihovi točni uzroci i mehanizmi uglavnom su nepoznati. Prethodne studije identificirale su nekoliko gena koji igraju ulogu u njihovom razvoju. Nuklearni distribucijski element 1 (NDE1) i nuklearni distribucijski element sličan 1 (NDEL1) su proteini nastali duplikacijom gena koji sudjeluju u staničnoj mitozi i neurološkom razvoju te su povezani s malformacijama mozga i neurološkim poremećajima. Iako su strukturno slični, njihove se patofiziološke funkcije značajno razlikuju.
Pristranost rijetkim kodonima je tendencija da se neki kodon koristi češće od drugih u određenoj vrsti, kada je riječ o onima koji kodiraju istu aminokiselinu. Kod ljudi, NDEL1 gen pokazuje veću učestalost korištenja rijetkih kodona u odnosu na NDE1 gen. Pokazalo se da rijetkost kodona utječe na brzinu translacije, savijanje proteina, kontrolu translacije i razine ekspresije proteina u većem broju istraženih genoma.
U diplomskom radu najprije sam istražila je li razlika u rijetkosti kodona između NDE1 i NDEL1 očuvana u više reprezentativnih vrsta rodova kralješnjaka. Zatim sam istražila može li zamjena kodona odgovornih za kodiranje NDE1 i NDEL1 ublažiti razlike u razinama ekspresije između ova dva proteina. U tu svrhu ispitivala sam NDE1 konstrukte kodirane kodonima bliskim onima koje koristi NDEL1 uz očuvanje točne aminokiselinske sekvence, i obrnuto. Na kraju sam fluorescentnom mikroskopijom ispitala gdje se unutar stanica eksprimiraju NDE1 i NDEL1 proteini divljeg tipa i onih s promijenjenim kodonima kako bi utvrdili postoje li očite razlike.
Pristranost za rijetke kodone kod NDEL1 ranije je uočena kod ljudi, a sada i kod primata i sisavaca koji nisu primati, što ukazuje da je razlika u pristranosti evolucijski očuvana u sisavcima. Naši rezultati pokazali su da je razlika u rijetkosti kodona u divljem tipu NDE1 i NDEL1 imala utjecaj na njihovu razinu ekspresije, pri čemu NDE1 pokazuje veću ekspresiju unutar stanica. Pri ispitivanju konstrukata sa zamijenjenim kodonima, postojala je indikacija da je ova razlika u ekspresiji poništena. Ovo opažanje sugerira da bi korištenje kodona koji se češće pojavljuju moglo potencijalno preokrenuti trend nižih razina ekspresije proteina, te da bi pristranost za rijetke kodone mogla biti djelomično odgovorna za različite funkcije dvaju proteina.
Keywords
chronic mental illness
NDE1
NDEL1
rare codon bias
protein expression
Keywords (croatian)
kronična mentalna bolest
NDE1
NDEL1
pristranost rijetkim kodonima
ekspresija proteina
Language english
URN:NBN urn:nbn:hr:193:006846
Study programme Title: Biotechnology in medicine Study programme type: university Study level: graduate Academic / professional title: magistar/magistra biotehnologije u medicini (magistar/magistra biotehnologije u medicini)
Type of resource Text
File origin Born digital
Access conditions Open access
Terms of use
Created on 2023-09-28 14:52:53