Yeast cleavage factor Hrp1 is a novel guard protein that surveils pre-mRNA 3’ processing
by Jing Li
Date of Examination:2022-03-22
Date of issue:2022-05-10
Advisor:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Heike Krebber
Referee:Dr. Oliver Valerius
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EnglishHrp1 is a component of the cleavage and polyadenylation complex (CPF-CF) for pre-mRNA cleavage and polyadenylation in S. cerevisiae. It specifically binds to a relatively conserved UA-rich domain called the efficiency-element (EE) upstream of the cleavage site to promote the accuracy and efficiency of pre-mRNA 3’ processing. Moreover, it functions in nonsense-mediated decay (NMD) and therefore commutes between the nucleus and the cytoplasm. Interestingly, it shares similarities with guard proteins that surveil mRNA processing. It contains two RNA-binding motifs and a typical SR/RGG (serine-arginine/arginine-glycine-glycine) rich domain. We propose that Hrp1 might also be a guard protein that surveils pre-mRNA cleavage and polyadenylation and have carried out a series of experiments to support this idea. We found that Hrp1 physically and genetically interacts with the mRNA export machinery Mex67-Mtr2 and directly contacts a component of the nuclear pore complex (NPC) named Mlp1, which is important for surveillance of mRNA export. Similar to the other guard proteins, overexpression of HRP1 is toxic to cells and retains mRNAs in the nucleus. Most strikingly, in comparison to the nuclear retention of faulty mRNAs in the exosome mutant rrp6Δ and the CPF-CF complex mutant cft2-1, these RNAs leak into the cytoplasm when functional Hrp1 is missing. In fact, we were able to show that 3’-elongated mRNAs reached the cytoplasm in the hrp1-1 cft2-1 double mutant with cell fractionation experiments. Moreover, we discovered that Hrp1 binds more faulty mRNAs in cft2-1 but recruits less Mex67, which is consistent with its function in nuclear retention. Interestingly, Hrp1 has lost physical interaction with its binding partner Rna14 in the CPF-CF complex in cft2-1. In this mutant, Rna14 is not incorporated into the CPF-CF complex anymore. Thus, we propose that Rna14 might be the trigger for Hrp1 mediated recruitment of Mex67 to mRNAs. In conclusion, our data reveal that Hrp1 is a novel guard protein that surveils the 3’ processing of pre-mRNAs.
Keywords: Hrp1; mRNA 3' processing; CPF-CF; guard protein; shuttling SR/RGG protein; Mex67; Mlp1; Rna14