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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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The GXXG motif in the KH domain of PNPase participates in RNA binding. (A) Sequence alignment of the KH domain of PNPase from different species shows that the GXXG motif is highly conserved. Secondary structure elements are shown on the top, with arrows indicating β-sheets and cylinders indicating α-helices. The PNPase-specific GXXG sequences are marked with a box. Sequences listed here include PNPase from Homo sapiens (Hs), Arabidopsis thaliana (At), Spinacia oleracea (So), E. coli (Ec) and S. antibioticus (Sa). (B) Sequence alignment of the KH domain in Rrp40 and Rrp4 exosome components shows that the GXXG motif was not conserved. Sequences listed here include components from the following species: Saccharomyces cerevisiae (Sc), H. sapiens (Hs), S. solfataricus (Ss) and Archaeoglobus fulgidus (Af). (C) EMSA assay showing that the hPNPase mutant G622D lost its RNA-binding activity in binding a 12-mer poly(A) ssRNA. (D) RNase activity assay showing that the hPNPase mutant G622D digested the 12-mer poly(A) RNA poorly as compared to wild-type PNPase.
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gkr1281-F6: The GXXG motif in the KH domain of PNPase participates in RNA binding. (A) Sequence alignment of the KH domain of PNPase from different species shows that the GXXG motif is highly conserved. Secondary structure elements are shown on the top, with arrows indicating β-sheets and cylinders indicating α-helices. The PNPase-specific GXXG sequences are marked with a box. Sequences listed here include PNPase from Homo sapiens (Hs), Arabidopsis thaliana (At), Spinacia oleracea (So), E. coli (Ec) and S. antibioticus (Sa). (B) Sequence alignment of the KH domain in Rrp40 and Rrp4 exosome components shows that the GXXG motif was not conserved. Sequences listed here include components from the following species: Saccharomyces cerevisiae (Sc), H. sapiens (Hs), S. solfataricus (Ss) and Archaeoglobus fulgidus (Af). (C) EMSA assay showing that the hPNPase mutant G622D lost its RNA-binding activity in binding a 12-mer poly(A) ssRNA. (D) RNase activity assay showing that the hPNPase mutant G622D digested the 12-mer poly(A) RNA poorly as compared to wild-type PNPase.

Mentions: A common feature of the KH domain is a GXXG motif which is located between two α-helices and responsible for nucleic acid binding (38). Structure-based sequence alignment of the KH domain of PNPases showed that the GXXG motif is highly conserved in PNPase from different species (Figure 6A). However, the GXXG motif is not conserved in Rrp40 and Rrp4 exosome subunits; instead, they contain another motif, GXNG, with unknown function located between two β-sheets (Figure 6B). The GXXG motif of the KH domain is involved in RNA binding in several proteins, such as NusA and ERA (39,40). In hPNPase, this GXXG motif is located in a β-turn region interacting directly with the bound RNA substrate in the hPNPase–RNA complex model (Figure 4C). We therefore hypothesized that the GXXG motif located inside the KH pore of PNPase is involved in RNA binding.Figure 6.


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)

The GXXG motif in the KH domain of PNPase participates in RNA binding. (A) Sequence alignment of the KH domain of PNPase from different species shows that the GXXG motif is highly conserved. Secondary structure elements are shown on the top, with arrows indicating β-sheets and cylinders indicating α-helices. The PNPase-specific GXXG sequences are marked with a box. Sequences listed here include PNPase from Homo sapiens (Hs), Arabidopsis thaliana (At), Spinacia oleracea (So), E. coli (Ec) and S. antibioticus (Sa). (B) Sequence alignment of the KH domain in Rrp40 and Rrp4 exosome components shows that the GXXG motif was not conserved. Sequences listed here include components from the following species: Saccharomyces cerevisiae (Sc), H. sapiens (Hs), S. solfataricus (Ss) and Archaeoglobus fulgidus (Af). (C) EMSA assay showing that the hPNPase mutant G622D lost its RNA-binding activity in binding a 12-mer poly(A) ssRNA. (D) RNase activity assay showing that the hPNPase mutant G622D digested the 12-mer poly(A) RNA poorly as compared to wild-type PNPase.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1281-F6: The GXXG motif in the KH domain of PNPase participates in RNA binding. (A) Sequence alignment of the KH domain of PNPase from different species shows that the GXXG motif is highly conserved. Secondary structure elements are shown on the top, with arrows indicating β-sheets and cylinders indicating α-helices. The PNPase-specific GXXG sequences are marked with a box. Sequences listed here include PNPase from Homo sapiens (Hs), Arabidopsis thaliana (At), Spinacia oleracea (So), E. coli (Ec) and S. antibioticus (Sa). (B) Sequence alignment of the KH domain in Rrp40 and Rrp4 exosome components shows that the GXXG motif was not conserved. Sequences listed here include components from the following species: Saccharomyces cerevisiae (Sc), H. sapiens (Hs), S. solfataricus (Ss) and Archaeoglobus fulgidus (Af). (C) EMSA assay showing that the hPNPase mutant G622D lost its RNA-binding activity in binding a 12-mer poly(A) ssRNA. (D) RNase activity assay showing that the hPNPase mutant G622D digested the 12-mer poly(A) RNA poorly as compared to wild-type PNPase.
Mentions: A common feature of the KH domain is a GXXG motif which is located between two α-helices and responsible for nucleic acid binding (38). Structure-based sequence alignment of the KH domain of PNPases showed that the GXXG motif is highly conserved in PNPase from different species (Figure 6A). However, the GXXG motif is not conserved in Rrp40 and Rrp4 exosome subunits; instead, they contain another motif, GXNG, with unknown function located between two β-sheets (Figure 6B). The GXXG motif of the KH domain is involved in RNA binding in several proteins, such as NusA and ERA (39,40). In hPNPase, this GXXG motif is located in a β-turn region interacting directly with the bound RNA substrate in the hPNPase–RNA complex model (Figure 4C). We therefore hypothesized that the GXXG motif located inside the KH pore of PNPase is involved in RNA binding.Figure 6.

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