Understanding mRNA fate regulation by RGG motif protein, Sbp1

Abstract

Cellular events that rely on translation regulation has been well established, however the molecular details of factors involved in bringing translation control remains inadequately explored. RNA binding proteins form an integral part of transcriptional and post-transcriptional gene regulation pathway. However, the principles that govern the activity of an RNA binding protein is poorly explored. In this thesis, a systematic investigation has been done to delineate the contribution of individual RRM domains and arginine methylation in the RGG motif of an RNA binding protein, Sbp1 towards its function. Chapter 3 demonstrated that role of arginine methylation of Sbp1 RGG motif towards its translation repression and decapping enhancing activity. Pull-down assays indicated that Sbp1 interaction with eIF4G1 decreases when the methylating enzyme, Hmt1 is absent or the Sbp1 RGG motif is deleted. We also learned that Sbp1 mono-methylation increases upon glucose starvation stress, which is known to cause global translation repression in yeast. Moreover, arginine methylation of Sbp1 was found to be crucial for driving decapping activators such as Dhh1 and Scd6 to RNA granules. Together, our results have established functional relevance of arginine methylation towards translation repression and decapping enhancing ability of RNA binding protein, Sbp1. Chapter 4 investigated the role of RRM domains of Sbp1 towards causing over-expression mediated growth defect and localizing to RNA granules. RRM domains are the most abundant RNA binding domain that harbor 6-8 amino acid consensus sequence involved in RNA binding. Our results have demonstrated that upon deleting both the RRM domains and not the RNP sequence, SBP1 over-expression mediated growth defect can be rescued. Moreover, ∆RRM 1+2 mutant of Sbp1 could not localize to RNA granules upon glucose starvation than wild-type. These observations suggest that Sbp1 RRM domains function via sequences outside the RNP motif, which is yet to be discovered. Chapter 5 describes novel genetic interaction of SBP1 with genes involved in Non-sense mediated mRNA decay (NMD) pathway. Over-expression mediated growth defect by SBP1 is augmented upon individual deletion of UPF1, UPF2 and UPF3. However, the augmented growth phenotype was not due to an increase in the protein level of Sbp1. Our study has established a link between translation repression and mRNA decay. To summarize, our study has identified: i) importance of arginine methylation of Sbp1 in regulating its function, ii) contribution of RRM domains of Sbp1 in causing over-expression mediated growth defect and localize to RNA granules upon stress and iii) genetic modulators of Sbp1 function. These studies have been done in budding yeast. Although, yeast does not display tissue level specificity observed in complex organisms, the molecular pathways are largely conserved. Principles that govern mRNA fate in yeast can form the basis for hypothesizing how certain factors might function in humans.