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Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association.

ABSTRACT: Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K+ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K+, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.

SUBMITTER: He GJ

PROVIDER: S-EPMC6430076 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Project description:We sought to determine the effect of TbPARN-1 overexpression on global mRNA steady-state levels in T. brucei. The mRNA expression profile of tet-induced TbPARN-1 OvEx procyclic cells was compared to the mRNA profile of control cells (expressing tet-induced TAP tag alone) by microarray analysis. Since overexpression of TbPARN-1 showed increased deadenylation activity in cytoplasmic extracts, overexpressing PARN-1 in vivo should result in more rapid deadenylation and decay of mRNAs targeted by PARN-1. Thus, mRNAs with lower steady state levels in PARN-1 OvEx cells can be considered likely targets for PARN-1-dependent deadenylation. Three different clones of the PARN-1 OvEx and Control cell lines were used for analysis. Each experiment was run in duplicate, with the dye-labeling reversed between duplicates. The T. brucei version 3 microarray chip (PFGRC) was used for this experiment. Each gene on the microarray was represented in duplicate, so for each experiment, mRNA levels were obtained from both points and averaged. As a control between arrays, we used a control probe set obtained from PFGRC. "The probe set consists of Cy3 and Cy5 end labeled 40-mer probes that are complementary to a set of 500 Arabidopsis thaliana 70-mer targets on PFGRC DNA microarrays. The UMSS is supplied as a ready to use mix of the Cy3 and Cy5 labeled oligonucleotides. A small aliquot is spiked into Cy-dye labeled nucleic acid samples just prior to hybridization on PFGRC microarrays. The UMSS produces a set of reference fluorescent signal intensities and ratios that are generated independent of the user-specific experimental samples. Since PFGRC intentionally targeted a heavy representation of Cy3 and Cy5 signals in the mid-range of detection, the Cy3 and Cy5 signal intensities and ratios generated by the UMSS should be highly reproducible. These signals serve as a reference point to judge the success of microarray experiments, and troubleshoot potential problems."

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Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association.

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