Abstract
It is now widely recognised that the human genome produces not only protein-coding messengers but also various transcripts with noncoding functions. Noncoding RNAs are involved in diverse cellular processes, including chromatin remodelling, transcriptional regulation, post-transcriptional processing, translation, and subcellular compartmentalisation. Many of these functions depend on RNAs' ability to recruit specific RNA-binding proteins (RBPs), particularly for transcripts involved in the assembly of large ribonucleoprotein complexes or membraneless compartments.Simple repeats of short nucleotide sequences, known as microsatellites or short tandem repeats (STRs), occupy a significant portion of the human genome. These elements are often genetically unstable and can contribute to the development of diseases. Notably, STR transcription can give rise to RNAs capable of multivalent recruitment of cognate RBPs, contributing to disease progression. The expansion of repeated GGGGCC hexanucleotides in the 5'-proximal part of the C9orf72 gene is a leading cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which affect motor and frontotemporal cortex neurons, respectively.
In Chapter 3 of this thesis, I conducted extensive analyses of the sense and antisense transcripts produced from repeat-expanded C9orf72 loci in patient-derived induced pluripotent stem cells (iPSCs) and motor neurons generated by iPSC differentiation in vitro. These experiments shed light on the expression dynamics, processing, cellular localisation, and aggregation properties of the repeat-containing transcripts. Moreover, my analyses uncovered a novel transcript isoform of C9orf72 that may inform the development of novel therapeutic approaches.
In Chapter 4, I investigated protein interactomes of the endogenous sense and antisense C9orf72 transcripts containing expanded repeats. To this end, I contributed to the development and optimisation of hybridisation proximity (HyPro) labelling, a new technology that can identify cellular neighbours of an RNA transcript of interest. I analysed the sense and antisense C9orf72 transcripts using this method, identified labelled proteins by mass spectrometry, and validated shortlisted hits from this screen by RNA-FISH and immunofluorescence.
In Chapter 5, I carried out bioinformatic analyses to systematically explore the potential of genetically normal STRs to be transcribed during neuronal differentiation. This uncovered STR- containing RNAs (STR-RNAs) that are differentially regulated in developing neurons. I then used RT-qPCR to validate some of these bioinformatics predictions in a newly developed system for doxycycline-inducible neuronal differentiation of iPSCs. I also analysed possible effects of STRs on splicing regulation.
In summary, my work provided new insights into the ALS/FTD pathology, facilitated the development of a new technology to study RNA-containing compartments, and began to uncover the possible functions of transcribed STR sequences in healthy cells.
| Date of Award | 1 Dec 2023 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Evgeniy Makeyev (Supervisor) & Benedikt Berninger (Supervisor) |