Global architecture of human poly(A)-specific ribonuclease by atomic force microscopy in liquid and dynamic light scattering

Abstract

Deadenylation is the initial and often rate-limiting step in the main pathways of eukaryotic mRNA decay. Poly(A)-specific ribonuclease (PARN) is a eukaryotic enzyme that efficiently degrades mRNA poly(A) tails. Structural and functional studies have shown that human PARN is composed of at least three functional domains, i.e. the catalytic nuclease domain and two RNA binding domains, the R3H and the RNA recognition motif (RRM), respectively. However, the complete structure of the full length protein is still unknown. We have investigated the global architecture of human PARN by atomic force microscopy (AFM) imaging in buffered milieu and report for the first time the dimensions of the full length protein at subnanometre resolution. The AFM images of single PARN molecules reveal compact ellipsoidal dimers (10.9 × 7.6 × 4.6 nm). The dimeric form of PARN was confirmed by dynamic light scattering (DLS) measurements that rendered a molecular weight of 161 kDa, in accordance with previous crystal structures of PARN fragments showing a dimeric composition. We discuss a putative internal arrangement of three functional domains within the full length PARN dimer.

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

“…5. This hypothesis is consistent with the previously AFM result, which showed that the full-length PARN has a compact globular architecture [13]. The interaction interface between CTD and the RNA-binding domains did not interfere with the binding sites of substrate and allosteric regulators [22] but enhanced domain stability against heat-induced inactivation (Fig.…”

“…[5]). However, little is known about CTD of PARN, which contains about 120 amino acid residues and is predicted to be intrinsically unstructured [13,14]. The existence of two PARN proteolytic fragments with the removal of CTD in Xenopus and HeLa cell extracts [24,25] implies that CTD may have regulatory roles in the cells.…”

“…1A). Besides these three well-defined domains, PARN also has a C-terminal domain (CTD) predicted to be intrinsically disordered by bioinformatics analysis [13,14]. PARN mainly exists as a homodimer in solutions, while it can also associate into larger oligomers via its R3H domain [15].…”

Activity and Function of Deadenylases

“…The major dimerization surface is formed by the nuclease domain, and mutations disrupting it strongly reduce enzymatic activity [41]. The C-terminal $130 amino acids are predicted to be unstructured but may contain a second dimerization surface [43]. As a member of the DEDD superfamily of 3 0 exonucleases described earlier [40,41,44,45], PARN degrades RNA from the 3 0 end, releasing 5 0 -NMPs.…”

Kiss your tail goodbye: The role of PARN, Nocturnin, and Angel deadenylases in mRNA biology

“…PARN exists as a dimer with the exonuclease domain acting to coordinate the two subunits [16]. The dimeric structure of PARN has been determined by X-ray crystallography of partial proteins [17], analytical ultracentrifugation [16], and in solution for the entire protein by atomic force microscopy and dynamic light scattering [18]. PARN requires divalent cations, particularly Mg 2+ , for optimal enzymatic activity [19].…”