Skip to main content

LUP Student Papers

LUND UNIVERSITY LIBRARIES

Developing a programmable mRNA pseudouridylation system to monitor gene-specific translation in human cells

Katsikas, Angelos (2023) MOBN03 20222
Degree Projects in Molecular Biology
Popular Abstract
A guide in the darkness; A system shedding light on mRNA pseudouridylation by PUS7

Proteins, the building blocks of life, are produced from messenger RNA (mRNA). mRNA is built by four ribonucleotides, namely adenine (A), guanine (G), cytosine (C) and uridine (U). Interestingly, RNA includes also chemically modified versions of the above nucleotides affecting many physiological processes. The most widespread of these modifications is pseudouridine (Ψ), which is a modified version of U. Ψ is introduced by specific cellular enzymes known as pseudouridine synthases (PUSs). PUS7 is one of these proteins and has key roles in development and disease by regulating the function of different RNA molecules. However, how pseudouridylation by PUS7... (More)
A guide in the darkness; A system shedding light on mRNA pseudouridylation by PUS7

Proteins, the building blocks of life, are produced from messenger RNA (mRNA). mRNA is built by four ribonucleotides, namely adenine (A), guanine (G), cytosine (C) and uridine (U). Interestingly, RNA includes also chemically modified versions of the above nucleotides affecting many physiological processes. The most widespread of these modifications is pseudouridine (Ψ), which is a modified version of U. Ψ is introduced by specific cellular enzymes known as pseudouridine synthases (PUSs). PUS7 is one of these proteins and has key roles in development and disease by regulating the function of different RNA molecules. However, how pseudouridylation by PUS7 controls the fate of RNA molecules coding for proteins involved in growth, differentiation and survival of human cells remains poorly understood.

To answer the question, we developed a novel system that allows PUS7 induced modification of specific U residues. CRISPR/Cas9 systems have been previously used to edit the sequence of DNA. These systems utilize a dCas9 that has lost its ability to cut DNA. dCas9 acts as a molecular compass that guides the modifying enzyme to a target site on DNA. Recently, a CRISPR/Cas13 system was discovered, where Cas13 recognizes and cuts RNA at specific sites. A dCas13d protein retains the ability to recognize RNA but is unable to cut it. Thus, we wondered whether we could achieve pseudouridylation at specific U nucleotides on RNA by engineering a “programmable” PUS7 enzyme. We successfully expressed PUS7-dCas13d in a human cell line. To study whether PUS7 could function properly when fused to dCas13d in human cells, we targeted PUS7-dCas13d to a region close to a U residue that is known to be modified by PUS7. We observed that PUS7-dCas13d was able to modify this U into Ψ. This indicates that fusing PUS7 to dCas13d does not affect its function and thus, our novel system can be used to induce pseudouridylation of any given U where PUS7 is normally able to deposit Ψ. However, since the rate of pseudouridylation was low, we still need to optimize some parameters of the system to increase the pseudouridylation rate at the targeted U.

Our results suggest that we have developed a functional system that allows us to study the impact of PUS7 driven pseudouridylation on mRNA. This will help unravel the function of this modification in molecular mechanisms active in development and disease, which will be beneficial for developing more effective therapies for diseases caused by abnormal pseudouridylation.

Master’s Degree Project in Molecular Biology 60 credits 2023 Department of Biology, Lund University

Supervisor: Cristian Bellodi, PhD
Division of Molecular Hematology, BMC, Faculty of Medicine, Lund University (Less)
Please use this url to cite or link to this publication:
author
Katsikas, Angelos
supervisor
organization
course
MOBN03 20222
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9140770
date added to LUP
2023-11-02 11:07:23
date last changed
2023-11-02 11:07:23
@misc{9140770,
  author       = {{Katsikas, Angelos}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Developing a programmable mRNA pseudouridylation system to monitor gene-specific translation in human cells}},
  year         = {{2023}},
}