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

Lin CL, Wang YT, Yang WZ, Hsiao YY, Yuan HS - Nucleic Acids Res. (2011)

Bottom Line: The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore.Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase.Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.

ABSTRACT
Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease that degrades specific mRNA and miRNA, and imports RNA into mitochondria, and thus regulates diverse physiological processes, including cellular senescence and homeostasis. However, the RNA-processing mechanism by hPNPase, particularly how RNA is bound via its various domains, remains obscure. Here, we report the crystal structure of an S1 domain-truncated hPNPase at a resolution of 2.1 Å. The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore. Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase. Our studies thus provide structural and functional insights into hPNPase, which uses a KH pore to trap a long RNA 3' tail that is further delivered into an RNase PH channel for the degradation process. Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

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Comparison of the S1 pore in exosomes and the KH pore in hPNPase. (A) The archaeal exosome (PDB entry 2JE6) has an S1 pore formed by the three S1 domains of Rrp4 (blue). The human exosome (PDB entry 2NN6) has a more open S1 pore formed by the S1 domains of Rrp4, Rrp40 and Csl4 (blue). In contrast, human PNPase has a KH pore formed by three KH domains (magenta). (B) Molecular surface representation of the archaeal exosome, human exosome and human PNPase structures show clearly differences in the arrangement of the domains that form the S1 pore and KH pore in exosomes and hPNPase, respectively. The color code is the same as in panel A: S1 domain in blue, KH domain in magenta, RNase PH domain in yellow, NTD domain (in Rrp40, Rrp4 and Csl4) in gray and CTD domain (in Csl4) in beige.
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gkr1281-F3: Comparison of the S1 pore in exosomes and the KH pore in hPNPase. (A) The archaeal exosome (PDB entry 2JE6) has an S1 pore formed by the three S1 domains of Rrp4 (blue). The human exosome (PDB entry 2NN6) has a more open S1 pore formed by the S1 domains of Rrp4, Rrp40 and Csl4 (blue). In contrast, human PNPase has a KH pore formed by three KH domains (magenta). (B) Molecular surface representation of the archaeal exosome, human exosome and human PNPase structures show clearly differences in the arrangement of the domains that form the S1 pore and KH pore in exosomes and hPNPase, respectively. The color code is the same as in panel A: S1 domain in blue, KH domain in magenta, RNase PH domain in yellow, NTD domain (in Rrp40, Rrp4 and Csl4) in gray and CTD domain (in Csl4) in beige.

Mentions: Previous structural analysis revealed the presence of an S1 pore for RNA binding in human and archael exosomes (29–31). Three Rrp4 (or Csl4) subunits form the S1 pore bound on the top of the hexameric ring in archaeal exosomes, whereas three RNA-binding proteins, Rrp40, Rrp4 and Csl4, associate to an open S1 pore in human exosomes (see Figure 3). In contrast, in hPNPase, the three KH domains form a KH pore situated on the top of the hexameric ring-like structure. The KH pore extends the central channel formed by the RNase PH domains and therefore is likely involved in the binding of RNA substrates, which are further delivered to the active site located within the central channel.Figure 3.


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

Lin CL, Wang YT, Yang WZ, Hsiao YY, Yuan HS - Nucleic Acids Res. (2011)

Comparison of the S1 pore in exosomes and the KH pore in hPNPase. (A) The archaeal exosome (PDB entry 2JE6) has an S1 pore formed by the three S1 domains of Rrp4 (blue). The human exosome (PDB entry 2NN6) has a more open S1 pore formed by the S1 domains of Rrp4, Rrp40 and Csl4 (blue). In contrast, human PNPase has a KH pore formed by three KH domains (magenta). (B) Molecular surface representation of the archaeal exosome, human exosome and human PNPase structures show clearly differences in the arrangement of the domains that form the S1 pore and KH pore in exosomes and hPNPase, respectively. The color code is the same as in panel A: S1 domain in blue, KH domain in magenta, RNase PH domain in yellow, NTD domain (in Rrp40, Rrp4 and Csl4) in gray and CTD domain (in Csl4) in beige.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351181&req=5

gkr1281-F3: Comparison of the S1 pore in exosomes and the KH pore in hPNPase. (A) The archaeal exosome (PDB entry 2JE6) has an S1 pore formed by the three S1 domains of Rrp4 (blue). The human exosome (PDB entry 2NN6) has a more open S1 pore formed by the S1 domains of Rrp4, Rrp40 and Csl4 (blue). In contrast, human PNPase has a KH pore formed by three KH domains (magenta). (B) Molecular surface representation of the archaeal exosome, human exosome and human PNPase structures show clearly differences in the arrangement of the domains that form the S1 pore and KH pore in exosomes and hPNPase, respectively. The color code is the same as in panel A: S1 domain in blue, KH domain in magenta, RNase PH domain in yellow, NTD domain (in Rrp40, Rrp4 and Csl4) in gray and CTD domain (in Csl4) in beige.
Mentions: Previous structural analysis revealed the presence of an S1 pore for RNA binding in human and archael exosomes (29–31). Three Rrp4 (or Csl4) subunits form the S1 pore bound on the top of the hexameric ring in archaeal exosomes, whereas three RNA-binding proteins, Rrp40, Rrp4 and Csl4, associate to an open S1 pore in human exosomes (see Figure 3). In contrast, in hPNPase, the three KH domains form a KH pore situated on the top of the hexameric ring-like structure. The KH pore extends the central channel formed by the RNase PH domains and therefore is likely involved in the binding of RNA substrates, which are further delivered to the active site located within the central channel.Figure 3.

Bottom Line: The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore.Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase.Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.

ABSTRACT
Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease that degrades specific mRNA and miRNA, and imports RNA into mitochondria, and thus regulates diverse physiological processes, including cellular senescence and homeostasis. However, the RNA-processing mechanism by hPNPase, particularly how RNA is bound via its various domains, remains obscure. Here, we report the crystal structure of an S1 domain-truncated hPNPase at a resolution of 2.1 Å. The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore. Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase. Our studies thus provide structural and functional insights into hPNPase, which uses a KH pore to trap a long RNA 3' tail that is further delivered into an RNase PH channel for the degradation process. Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

Show MeSH
Related in: MedlinePlus