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.

The Intrinsically Disordered C-Terminal Domain Triggers Nucleolar Localization and Function Switch of PARN in Response to DNA Damage

“…Most factors affect the fluorescence intensity in a non-specific way, except for K + , which showed an unusual enhancement in a K + concentration-dependent manner with a K d value of 7 mM (Figure S2). This phenomenon was similar to that observed in full length PARN [34].…”

“…The CTD has been predicted to be intrinsically disordered based on in silico analysis of the amino acid sequence [30,31]. Previous biochemical and biophysical analysis indicates that the CTD contributes little to PARN structural integrity and catalysis [32,33], while playing a role in PARN stability and self-association [34]. The cytoplasm localization of proteolytic fragments of Xenopus PARN, which lacks the CTD, and human PARN, which lacks the CTD as well as half of the RRM, implies that the CTD contains the nuclear localization sequence (NLS) [35,36].…”

Deorphanizing Caspase-3 and Caspase-9 Substrates In and Out of Apoptosis with Deep Substrate Profiling

“…98,99 PARN possesses three well-structured RNA-binding domains [catalytic nuclease domain, an R3H domain, and an RNA recognition motif (RRM) domain] 100 and an intrinsically disordered C-terminal domain (CTD) (Figure 6A). 101,102 From our N-terminomics analyses, PARN was observed to be a substrate of both caspase-3 and -9, in which both caspases cleaved PARN in the CTD at the same site, D595 (Table 1 and Figure 6A). These results mirror our immunoblotting analysis (Figure 6B).…”

Poly (A)-specific ribonuclease (PARN): More than just “mRNA stock clearing”

“…PARN is unique among ribonucleases due to its unusual ability to interact with the 5' cap end and poly(A) tail of mRNAs [13][14][15][16][17]. The interaction of PARN with the 5'-cap of mRNA is thought to enhance the rate of mRNA degradation [18].…”

“…PARN belongs to the DEDD (Death effector domain containing DNA binding) family of nucleases, which binds to a divalent metal ion [18] and consists of R3H domain and the RRM domain [19] (Figure 4). A 54-kDa fragment of the total 74-kDa poly(A)-specific ribonuclease is an oligomeric, processive and cap interacting poly(A) specific 3' exonuclease [15], while the function of other 20KDa fragment is not known.…”