title: Investigating the importance of the SAF-A mesh and RNA recycling on transcription
Creators: name: Stocks, Jon
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: SAF-A
subject: RNA gel
subject: Transcription
subject: Neurodevelopmental disorder
subject: XRN2

description: Chromatin has a complex three-dimensional structure that enables precise transcriptional regulation. The majority of newly synthesised RNA is retained in the nucleus, but its role there is frequently overlooked. However, from recent studies it is becoming increasingly apparent that this RNA might be important for chromatin structure organisation. It has previously been shown that nuclear RNA associates with Scaffold Attachment Factor A (SAF-A), also known as HNRNPU, and forms a dynamic nuclear mesh that decompacts chromatin at active gene loci. Furthermore, we have shown that this mesh is hydrated in the aqueous environment of the nucleus creating a gel-like structure that can alter nuclear viscosity. A recently identified condition, HNRNPU-related neurodevelopmental disorder, is largely associated with haploinsufficiency of the protein and is characterised by developmental delay and intellectual disability. Despite having a significant influence on chromatin structure and mutations having a clear patient phenotype, no steady-state change in transcription is observed when SAF-A is knocked down. The aim of my thesis is to identify if the SAF-A RNA gel, rather than being associated with the maintenance of transcription, is involved in establishing a permissive environment for transcription initiation. I hypothesised that as transcription is activated the initial transcripts associate with SAF-A forming a gel that concentrates transcription machinery and decompacts the gene. This would result in an increase in the accessibility of the gene for transcription factors and RNA polymerases permitting a higher level of transcription. I used heat shock to substantially activate transcription and investigate the dependence of this transcriptional upregulation on SAF-A and its related proteins. I found that depletion of SAF-A reduced the extent of transcription of genes activated by the heat shock response ,including those involved in neuronal activity. Gene decompaction or recruitment of transcriptional machinery through a protein-RNA gel may be involved in enabling a rapid and increased induction in transcription in response to a stimulus. This may also explain the association between SAF-A mutations and neurodevelopmental conditions with SAF-A haploinsufficiency resulting in the inability to drive the transcriptional upregulation of genes involved in the development of the brain. I also investigated the impact of SAF-A associated proteins on transcription and the SAF-A gel. I discovered that, despite their sequence similarity and shared domains, depletion of the SAF-A orthologues, HNRNPUL1 and HNRNPUL2, had different effects on transcription, with knockdown of either resulting in large-scale transcriptional dysregulation in standard growth conditions. I also found that HNRNPUL1 is required to repress gene activity after heat shock whilst HNRNPUL2 is required to repress the transcription of these genes in non-heat shock conditions. This contributes to our understanding of how RNA binding proteins collaborate to precisely control transcription after a stimulus. Finally, I showed that the SAF-A associated exoribonuclease, XRN2, influenced the properties of SAF-A by modifying the amount of RNA in the SAF-A gel and a concomitant change in the clustering of SAF-A. This led me to investigate whether XRN2 is involved in the process of ribonucleotide recycling, a process in which RNA is degraded and the nucleotides rephosphorylated, enabling their reuse. I developed an assay that demonstrated that ribonucleotide recycling occurs in multiple cell lines, although the dependency of XRN2 on this process differs between cell types. This is a first step in identifying whether a complex of proteins is localised at active genes to rapidly recycle RNA, providing a concentrated pool of nucleotides to enable efficient transcription.