Structural Basis of m7GpppG Binding to Poly(A)-Specific Ribonuclease

Abstract

Poly(A)-specific ribonuclease (PARN) is a homodimeric, processive, and cap-interacting 3' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. The crystal structure of a C-terminally truncated PARN in complex with m(7)GpppG reveals that, in one subunit, m(7)GpppG binds to a cavity formed by the RRM domain and the nuclease domain, whereas in the other subunit, it binds almost exclusively to the RRM domain. Importantly, our structural and competition data show that the cap-binding site overlaps with the active site in the nuclease domain. Mutational analysis demonstrates that residues involved in m(7)G recognition are crucial for cap-stimulated deadenylation activity, and those involved in both cap and poly(A) binding are important for catalysis. A modeled PARN, which shows that the RRM domain from one subunit and the R3H domain from the other subunit enclose the active site, provides a structural foundation for further studies to elucidate the mechanism of PARN-mediated deadenylation.

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

“…These surprising results yielded from biophysical and biochemical methods were confirmed by the structures of the PARN RRM complexed with a cap analogue, resolved by multidimensional NMR methods [36] and crystallography [37,38] (Fig. 1d).…”

“…Here we have used atomic force microscopy (AFM [39]) in liquid for imaging of immobilized proteins, as well as dynamic light scattering (DLS) measurements and gel filtration experiments in solution to find estimates of the protein shape. We report for the first time the dimensions of the complete PARN molecule, which previously has not been possible to obtain neither by crystallographic studies using truncated versions of PARN [35,38] (Fig. 1) nor from solution studies [40].…”

“…13 with the fact that all known PARN RRM structures resolved by NMR or crystallography [36][37][38] are also divergent, strongly influenced by the exact amino acid sequence of the protein fragment, buffer composition and the crystallographic contacts. The observed PARN RRM structures are thus highly sensitive to the overall protein context.…”

“…Reconstruction of the hypothetical full length PARN molecule based on two fragmentary crystal structures [38] was the first speculative approach to visualization of putative arrangement of the functional domains within the dimer. However, that reconstruction kept the empty space in the centre of the molecule, as it was in the apo-structure of the nuclease and the R3H domains of PARN [35].…”

FXR1a-associated microRNP: A driver of specialized non-canonical translation in quiescent conditions

“…[83][84][85] Biochemical and structural studies revealed that PARN binds to the 7-methylguanosine cap (m 7 G) of mRNAs via specific tryptophan residues. [86][87][88][89] Cap binding enhances the deadenylation activity of PARN. 83,89 PARN is localized to both the nucleus and cytoplasm and is known to play a role in gene expression regulation.…”