AbstractRNA transcript abundance is largely decided by a careful balance between the
transcription and degradation of any given gene’s transcripts. RNA stability plays a
critical role in the availability of an RNA species, and the time period over which it is
able to elicit its functions and roles. The majority of the literature on regulation of RNA
activity by degradation focuses on mRNAs, with the assumption that their role is to be
translated into a functional protein, as described by the central dogma. Increasingly
though, non-coding RNAs have been recognised as crucial to the normal function of
biological organisms. The roles of RNA species such as miRNAs in controlling gene
expression are now relatively well understood, as are the molecular mechanisms by
which they bind to and regulate RNAs.
In contrast, long non-coding RNAs (lncRNAs) are a very poorly understood (and
arguably, defined) category of RNAs. However, they clearly have their own crucial roles
to play, as this relatively recently discovered RNA species regulates gene expression in
diverse ways, encodes small biologically relevant peptides, and has been involved in a
large variety of important biological functions. Importantly, an increasing number of
lncRNAs have also been associated with a range of human diseases, including
neurodegenerative pathologies and cancer. The degradation of lncRNAs requires
significant study, in order to bring understanding of this key regulatory step of a crucial
class of transcripts up to the level of that of the rest of the transcriptome.
The aim of this thesis is to investigate the degradation of lncRNAs by the
exoribonucleases Pacman and Dis3L2, in Drosophila melanogaster. Within this
overarching aim, several smaller goals arise. Firstly, this thesis investigates whether
certain lncRNAs are specifically and significantly degraded by Pacman and Dis3L2, as is
seen with canonical RNAs. By examining previous RNA sequencing data from
experiments carried out on exoribonuclease deficient Drosophila (both in vivo, and in
Drosophila derived cell lines,) it was possible to identify promising candidates for
lncRNAs with significantly altered abundance in the absence of either Pacman or Dis3L2.
This existing work was validated with qPCR, proving the principle of specific regulation
of lncRNAs by Pacman and Dis3L2.
Following this, an experiment was designed and carried out to examine the role of the
translating ribosome in this degradation. Existing work has shown the ribosome to be
associated with XRN1 and Pacman in humans and Drosophila respectively, and Dis3L2
has also been shown to associate with the ribosome in humans, although whether this
occurs in Drosophila is unclear. By using the powerful technique polyribosome
sequencing (poly-ribo-seq), on exoribonuclease deficient Drosophila samples, this work
has identified a preliminary set of lncRNAs that appear not only to be specifically
regulated by Pacman and Dis3L2, but also undergoing translation, indicating the
presence of small open reading frames (smORFs) within the lncRNA genes.
Ongoing work will validate not only the upregulation of these transcripts in the absence
of the relevant exoribonuclease, but also the putative smORF from which a peptide is
likely produced. Following this, it will investigate whether a block in transcription
eliminates the differential abundance of these transcripts in the absence of Pacman or
Dis3L2. This work then, identifies an initial subset of lncRNAs regulated by Pacman and
Dis3L2, and shows several of them to be actively translated, identifying novel peptides,
potentially of biological significance (given their active translation and specific
degradation). With the completion of ongoing work, this project will also elucidate
whether their translation is important to the degradation of these transcripts.
|Date of Award||Feb 2021|
|Supervisor||Sarah Newbury (Supervisor)|