Crystal structure of human polynucleotide phosphorylase: insights into its domain function in RNA binding and...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease involved in RNA degradation and mitochondrial RNA import.
• Its precise RNA-binding and processing mechanisms, particularly domain interactions, remain largely unknown.
• hPNPase regulates critical cellular processes like senescence and homeostasis.

Purpose of the Study:

• To elucidate the structural basis of RNA binding and processing by human polynucleotide phosphorylase (hPNPase).
• To investigate the roles of different domains, specifically the S1 and KH domains, in hPNPase function.
• To provide a mechanistic understanding of how hPNPase interacts with and degrades RNA.

Main Methods:

• X-ray crystallography to determine the structure of an S1 domain-truncated hPNPase at 2.1 Å resolution.
• Biochemical assays to assess RNA binding and degradation.
• Site-directed mutagenesis to probe the function of specific motifs and domains.

Main Results:

• The crystal structure reveals a hexameric ring-like assembly of RNase PH domains capped by a trimeric KH pore.
• The S1 domain is not essential for RNA binding.
• The conserved GXXG motif within the KH domain is crucial for direct RNA binding.
• hPNPase utilizes the KH pore to capture long RNA 3' tails for subsequent degradation by the RNase PH channel.
• Short 3' tailed RNAs are transported but not degraded.

Conclusions:

• The KH pore is the primary site for initial RNA 3' tail capture in hPNPase.
• The RNase PH domains facilitate the degradation of captured long RNA tails.
• hPNPase exhibits differential processing of RNA based on 3' tail length, impacting cellular RNA homeostasis.