Investigating the functions of scaffold attachment factor B (SAFB) proteins following cellular stress

Abstract

Scaffold Attachment Factor B1 (SAFB1) and SAFB2 are RNA and DNA binding proteins with roles in gene expression, DNA repair and the stress response. SAFB1 was identified first and has been the most intensely studied and the high degree of similarity between the two proteins has led to difficulty in elucidating their individual roles. In this study I have used advanced genetic techniques and SAFB1 and SAFB2 specific antibodies to discriminate the functions of the individual family members. Specifically, experiments have been conducted to investigate: (i) the specific roles of SAFB1 and SAFB2 proteins in the stress response,(ii) how SAFB protein function is regulated by post-translational modification; (iii) what the SAFB protein binding partners. Immunocytochemistry using SAFB1 and SAFB2 specific antibodies revealed for the first time that SAFB2 is recruited to nuclear stress bodies (nSBs) following heat stress (HS), co-localising with HSF1 in 85% of cells displaying canonical HSF1+ nSBs, whereas SAFB1 co-localised with HSF1 in only 6.7% of these cells. SAFB2 also co-localised with the HS induced lncSATIII RNA. SAFB2 knockdown in SH-SY5Y cells led to an increase in the expression lncSATIII transcripts following heat stress, an effect that was not seen with either SAFB1 knockdown or double SAFB1/SAFB2 knockdown. These findings suggest that SAFB2 rather than SAFB1 as previously proposed plays an important in nSB formation and regulation of the stress response. Global methylation inhibition strongly decreased the formation of canonical and secondary HSF1 nSBs following HS, increased large SAFB1 aggregate formation, and had no effect on SAFB2 distribution. Meanwhile, a PRMT5 inhibitor (inhibition of symmetric di-methyl arginine) significantly increased the formation of canonical HSF1 nSBs but produced no effect on the distribution of any SAFB family member. Moreover, SAFB1 and SAFB2 mutants containing impaired sites of SUMOylation and arginine methylation showed a delay in their recruitment to HSF1 nSBs during HS. Finally, tandem mass tagging mass spectrometry proteomic analysis of proteins coimmunoprecipitated with SAFB1 and SAFB2 was used to identify novel interacting partners for SAFB1 and SAFB2 proteins. A number of novel interaction partners were identified and as our laboratory is interested in miRNA function particular interest was raised by the observation that both SAFB1 and SAFB2 both co-immunoprecipitated with Ago2. Ago2 is key regulator of miRNA mediated silencing and is a novel interacting partner of SAFB proteins not previously reported in the literature. Furthermore, Ingenuity Pathway Analysis (IPA) was performed to explore the cellular processes regulated by SAFB and its interaction partners. Analyses also showed that the interaction partners of both SAFB1 and SAFB2 proteins were involved in miRNA processing, eIF2/ISR signalling, the unfolded protein response and in Huntington's Disease signalling, and these findings are discussed more fully in the thesis. Together, these data reinforce a key role of SAFB1 and SAFB2 in regulating critical cellular processes such as miRNA processing, cellular adaptation to stress and neurological disease development.

Date of Award	28 Sept 2021
Original language	English
Awarding Institution	University of Bristol
Sponsors	Brazilian Higher Education Funding Agency (CAPES)
Supervisor	James B Uney (Supervisor) & Liang-Fong Wong (Supervisor)