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Title: Characterisation of cytoplasmic lncRNA interactions with translation machinery during human neuronal differentiation
Author: Douka, Aikaterini
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2020
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Long non-coding RNAs (lncRNAs) are non-coding transcripts longer than 200nt, many of which are capped, spliced and polyadenylated, like mRNAs. However, lncRNAs exhibit more tissue and developmental-stage specificity than mRNAs and are particularly enriched in the nervous system. Several lncRNAs contribute to neuronal differentiation and lncRNA mis-regulation is implicated in neurological disorders. Whilst nuclear functions of lncRNAs are extensively studied, less is known about their cytoplasmic functions, even though many are localised there. LncRNAs contain small Open Reading Frames (smORFs) a small proportion of which show evidence of active translation, resulting in small peptide synthesis in S. cerevisiae, D. melanogaster, M. musculus and H. sapiens. However, these translation events remain poorly understood. This thesis aims to characterise the interactions of cytoplasmic lncRNAs with the translation machinery and investigate their coding potential, using Poly-Ribo-Seq, during the early stages of human neuronal differentiation. Differentiation of human SH-SY5Y cells with retinoic acid resulted in a significant reduction of active translation, revealed by polysome profiling. To determine the importance of lncRNA-polysome interactions in differentiation, Poly-Ribo-Seq was performed in undifferentiated and differentiated SH-SY5Y. Upon neuronal differentiation 178 lncRNAs are upregulated and 100 downregulated, 71% and 58% of which are associated with polysomes, respectively. Additionally, a dynamic interaction pattern between lncRNAs and polysomes was identified during differentiation, validated by RT-qPCR of specific cytoplasmically enriched lncRNAs across sucrose gradients. LncRNAs of Control and differentiated cells contain actively translated smORFs. 45 translated lncRNA smORFs were identified by Poly-Ribo-Seq. One of these is LINC01116 smORF, encoding for an 87aa putative peptide. Its translation was validated by FLAG-tagging assays in SH-SY5Y and HEK293 cells. It exhibits cytoplasmic distribution with distinct localisation in cell membrane protrusions. LINC01116 expression is upregulated 4-fold upon differentiation. LINC01116 knockdown results in significant reduction of neurite length and neuronal marker MOXD1 in differentiated cells indicating it contributes to neuronal differentiation.
Supervisor: Aspden, Julie ; Deuchars, Jim ; Whitehouse, Adrian Sponsor: University of Leeds
Qualification Name: Thesis (Ph.D.) Qualification Level: Thesis
EThOS ID:  DOI: Not available