Functional characterization of MKT1 and PBP1 proteins in Trypanosoma brucei

In the cytoplasm of a eukaryotic cell, the mRNAs are stored, translated and degraded. The balance between these processes is believed to be determined by multiple interactions between the mRNA and various RNA binding proteins. These interactions in turn are influenced by the gene regulation in response to various changes in the cellular environment. In T. brucei, the regulation of gene expression operates at the posttranscriptional level. ZC3H11, a CCCH zinc finger protein in T. brucei is known to specifically bind and stabilize the heat shock mRNAs. Through affinity purification, ZC3H11 was found to interact with MKT1 and PBP1. These proteins caught our interest, as their orthologues are known to exist and interact in yeast. Also, Ataxin-2, the orthologue of PBP1 in mammals is covered in various other studies, owing to its medical relevance. Furthermore, in both yeast and mammals, PBP1 was shown to interact with Poly (A) binding protein (PABP). During my PhD, I tried to decipher the role of MKT1 and PBP1 in T.brucei. I verified the interaction of both these proteins and found them to be essential for survival of bloodstream form of T.brucei. Through an extensive yeast two-hybrid analysis, I could show that both PBP1 and MKT1 are necessary for the function of ZC3H11; and PBP1 also interacts with Poly (A) binding protein (PABP). Thus, through my study, I propose a mechanism for action of ZC3H11 where it stabilizes its target chaperone-encoding mRNA by recruiting MKT1-PBP1 complex, which in turns interacts with PABP. The PABP then facilitates mRNA circularization by tethering the 3’ end with the 5’cap through its interaction with eIF4E. Additionally, through interaction partner search by MKT1 yeast two-hybrid library screen, it was found that MKT1 binds to a fair number of RNA binding proteins, some of which also contain a conserved HNPY motif towards their C-terminus. I then validated the importance of this motif in ZC3H11 function and also showed that its presence is sufficient for interaction of MKT1 with CFB1D (another interacting partner of MKT1, identified in the screen). I anticipate that the finding and mechanism proposed for ZC3H11 function, through this study, could perhaps be applicable to other RNA binding proteins, which interact with MKT1.Moreover, I could also show that just like in yeast, MKT1 and PBP1 both co-associate with the translating polyribosomes and PBP1 also relocates to stress granules after nutrient starvation. This is consistent with both these proteins playing roles in transcript regulation following cellular stress. Overall, through this study I suggest that trypanosome MKT1 acts as a ‘hub’ assisting in posttranscriptional regulation of various transcripts. I propose that MKT1 confers this role by binding directly or indirectly to a variety of RNA binding proteins thereby upgrading the ‘one transcript-one regulator’ models for gene regulation