Role of La related protein Slf1 in translation regulation during oxidative stress in yeast

Abstract

Protein synthesis is a complex biological process that is tightly controlled, especially during times of stress. While most protein synthesis ceases during stress, the production of a few proteins that are essential to cell survival is maintained. In this context, Slf1, an RNA binding protein of the LARP family, has been shown to promote the translation of particular RNAs that are essential to cellular oxidative stress responses to reactive oxygen species (ROS). When Slf1 is removed cells are hypersensitive to ROS. They fail to induce the translation of key antioxidant enzymes that are transcriptionally upregulated in response to the stress. Here experiments were done to investigate how Slf1 promotes timely translation of these antioxidant enzymes so that cells adapt, overcome the imposed stress and so continue to grow and divide. Chapter 3 describes investigations into translation initiation and its control. Early steps are typically major control points of most biological processes, including protein synthesis. All investigations demonstrated no clear impact of loss of Slf1 on translational control in response to ROS. ROS induces translational repression via phosphorylation of eIF2 and this was unchanged in SLF1 deletion cells. The interaction of the cap-binding protein eIF4E with Slf1-target mRNAs or with other initiation factors tested was similarly not impacted by SLF1 deletion.Moreover, a polysome fractionation-qPCR approach showed that ribosome recruitment onto selected target and non-target RNAs was not attenuated in the SLF1 deletion strain. Together these observations corroborate that Slf1 does not likely significantly impact the translation initiation process during stress adaptation. Chapter 4 reports results of investigations into impact on translation elongation and termination. This work extended findings of a PAR-CLIP study performed by Dr. Jennings that showed that Slf1 binds within ORFs, showing Slf1 might have role in later stages of translation. Ultimately, this work pinpointed a role for Slf1 in preventing ribosome frameshifting during translation elongation. Bioinformatics analysis of the CLIP data showed the binding sites of Slf1 coinside with peaks of ribosome monosome footprints as well as disome (collided ribosome) footprints. Interestingly, disome profiling and western blot analysis reveal that Slf1 binds both monosomes and collided ribosomes. Collided ribosome levels rise during ROS, but not in SLF1 deletion cells suggesting Slf1 stabilizes disomes. As ribosome stalls and collisions can promote ribosome frameshifting, programmed frameshifting reporters were used and it was found that SLF1 deletion significantly enhances both + 1 and - 1 ribosomal frameshifting highlighting its role in preventing aberrant ribosomal frameshift. The findings of this study are discussed in Chapter 5 with the development of an explanatory model suggesting that by maintaining translation elongation fidelity Slf1 orchestrates the translation of antioxidant RNAs, preventing ribosome collisions and ensuring correct protein synthesis to enhance cell survival during oxidative stress.

Date of Award	1 Aug 2024
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Graham Pavitt (Supervisor) & Martin Pool (Supervisor)