The role of long non-coding RNAs at the onset of haematopoietic commitment

Abstract

Mammalian genomes are promiscuously transcribed and produce several classes of RNAs, a large proportion of which are non-protein coding. Only a few such non-coding transcripts have been functionally characterised. In this thesis, the potential involvement of non-coding RNAs is investigated in the context of early haematopoiesis. To do this, an in vitro mouse embryonic stem (ES) cells model of haematopoietic development was adopted. This involved differentiating ES cells along the mesoderm lineage, from which the common endothelial and haematopoietic precursor, haemangioblast, was derived. The process of differentiating mESCs to blood progenitors was carried out over a 3 day time-course with cells harvested at every 12 hour interval. Exon microarray and RNA-sequencing (seq) screens of the time- course followed this. From these screens, annotated and unannotated, putative, non-coding loci were found to be differentially expressed, some of which correlated in expression to protein-coding genes with known involvement in early haematopoietic development. Specifically, from the exon microarray analysis a novel transcript was identified, with little or no predicted coding potential, antisense to the 3'UTR of Sall4, a key transcription factor required for ES cell self-renewal. Given the position of Sall4-AS relative to its sense, protein-coding partner, it was hypothesised that Sall4-AS could be masking miRNA binding sites. However, knockdown and overexpression studies did not support the proposed antisense-mediated regulatory mechanism, suggesting that Sall4-AS could be acting in a different cellular context or through a different regulatory mechanism. From the RNA- seq screen, differentially expressed long intergenic non-coding (linc) RNAs were identified, of which two candidates, Meg3 and Igf2as, were chosen for experimental validation. Both transcripts were knocked down in haematopoietic differentiation time-course cultures, of which Meg3 knockdown did not appear to affect the immuno-phenotype of the differentiating cells, whereas Igf2as knockdown caused transient immuno- phenotypic changes. Put together, these results show that despite the experimental challenges associated with dissecting the functions and mechanisms of ncRNA transcripts, they are differentially expressed in the adopted in vitro model of early haematopoiesis and are likely to be critical regulators of the process.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Crispin Miller (Supervisor) & Valerie Kouskoff (Supervisor)