Elucidating the diverse functions of RGG-motif containing proteins

Abstract

One year embargo upto 20/2/2026

RGG-motif containing proteins represent one of the major subsets of RNA-binding proteins characterized by the presence of RGG/RG repeats. These proteins have been associated with a wide array of functions, including transcription and translation regulation. Scd6 and Sbp1 are two such proteins that function as translation repressors and decapping activators. Scd6 (Suppressor of Clathrin Deficiency) was identified in a multigene and multicopy screen that suppressed clathrin deficiency-induced growth defect. This observation led us to test the possibility of Scd6 and other RGG-motif containing proteins working as suppressors of CHC1 deficiency. The first part of our study identifies SCD6 as a necessary and sufficient gene in a multi-gene construct SCD6 that suppresses clathrin deficiency. We also identify another RGG-motif protein-encoding gene, PSP2, as a novel suppressor of clathrin deficiency. Three other RGG-motif protein-encoding genes, SBP1, DED1, and GBP2, do not suppress clathrin deficiency. DHH1, a DEAD-box RNA helicase with translation repression activity, also fails to rescue clathrin deficiency. Further experimental analyses shed some light on the molecular details of the suppression activity of Scd6 and Psp2. Strikingly, Scd6 and Psp2 suppression activity depend on active TORC1, and Psp2 overexpression increases the abundance of ubiquitin-conjugated proteins in Chc1-depleted cells. Based on our results using SCD6 and PSP2, we identify a novel role of RGG-motif containing proteins in suppressing clathrin deficiency. Many RGG-motif proteins are known to localize and regulate the dynamics of membraneless structures called RNP condensates. These structures are the site of mRNA degradation and storage, thereby having an essential physiological role in the gene expression pathway. Scd6 and Sbp1 can localize to cytoplasmic RNP condensates in response to oxidative stress induced by sodium azide. Transcriptome-wide effects of these proteins and of sodium azide remain unknown. In the second part of the thesis, we have sequenced the transcriptome of the: a) wild type strain under unstressed and sodium azide stress, b) Δscd6 and Δsbp1 strains under unstressed and sodium azide stress. Transcriptome analysis identified the altered abundance of many transcripts belonging to stress-responsive pathways, further validated by qRT-PCR results. The abundance of several transcripts was altered in Δscd6/Δsbp1 under normal conditions and upon stress. Overall, this study provides critical insights into transcriptome changes in response to sodium azide stress and the role of RGG-motif proteins in these changes. Apart from their physiological roles, many RBPs are the major contributors to various neurodegenerative diseases (ND). For instance, RBPs like FUS, TDP43, TAF15, and EWSR1 have been associated with Amyotrophic Lateral Sclerosis (ALS), which is a progressive and ultimately fatal neurodegenerative disease with no cure to date. Pathologically, the key features of ALS include loss of upper and lower motor neurons and denervation changes within muscles. One of the other hallmarks of ALS and associated diseases is the mislocalization of various proteins from the nucleus to the cytoplasm and the formation of toxic cytoplasmic aggregates. The ability of these proteins to phase-separate into dynamic RNP condensates orchestrates their accumulation into pathological aggregates in response to specific stimuli and/or mutation. Research in previous years has shed some light on the importance of RGG-motif containing proteins in assembling these structures. A recent report from our lab described the role of an IDR containing protein from Saccharomyces cerevisiae, Sbp1, in the disassembly of P-bodies, a type of cytoplasmic RNA condensate. Interestingly, it was also noted that deletion of Sbp1 aggravated the EWSR1-mediated toxicity in a yeast model of ALS. The observations paved the way for our third part to address the possible role of RGG-motif containing proteins in the dissolution of disease-relevant condensates. These observations are extended to check the potential role of Sbp1 in mitigating the toxicity of aggregate-prone proteins, TDP43, and FUS. In this direction, we have assessed the role of Sbp1 in affecting the aggregation and toxicity of proteins implicated in ALS and identified a novel role that can have therapeutic potential. Overall, our study identifies novel functions of three RGG-motif containing proteins in different cellular pathways. This highlights the wide array of functions that can be performed by these proteins, apart from their canonical role of translation regulation, in both physiological and pathological contexts.