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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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Crystal structure of ΔS1 hPNPase. (A) The monomeric structure of ΔS1 hPNPase comprises two RNase PH domains (blue and yellow), an α-helical domain (green) and a KH domain (magenta). Two citrate ions (orange) bound near the active site are displayed in stick models. (B) Side view and top view of the trimeric ΔS1 hPNPase which assembled into a ring-like structure with a central channel. The three KH domains are located on the top of the ring, forming a novel RNA-binding KH pore. (C) Superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K, magenta) with those of S. antibioticus PNPase (PDB entry 1E3P, yellow) and E. coli PNPase (PDB entry 3CDI, gray). The RNase PH and α-helical domains matched well; however, the KH domain in hPNPase (marked by a dashed black oval) does not superimpose with the poly-alanine chain in S. antibioticus PNPase (marked by a dashed red oval). (D) Two citrate ions (stick models in orange) bind at the active site in the second RNase PH domain in hPNPase. (E) Trimeric and monomeric full-length structural models of hPNPase generated by superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K) with those of S. antibioticus PNPase (PDB entry 1E3P) and S1 domain of E. coli PNPase (PDB entry 1SRO). Blue, magenta and yellow represent S1 domain, KH domain and RNase PH domain, respectively.
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gkr1281-F2: Crystal structure of ΔS1 hPNPase. (A) The monomeric structure of ΔS1 hPNPase comprises two RNase PH domains (blue and yellow), an α-helical domain (green) and a KH domain (magenta). Two citrate ions (orange) bound near the active site are displayed in stick models. (B) Side view and top view of the trimeric ΔS1 hPNPase which assembled into a ring-like structure with a central channel. The three KH domains are located on the top of the ring, forming a novel RNA-binding KH pore. (C) Superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K, magenta) with those of S. antibioticus PNPase (PDB entry 1E3P, yellow) and E. coli PNPase (PDB entry 3CDI, gray). The RNase PH and α-helical domains matched well; however, the KH domain in hPNPase (marked by a dashed black oval) does not superimpose with the poly-alanine chain in S. antibioticus PNPase (marked by a dashed red oval). (D) Two citrate ions (stick models in orange) bind at the active site in the second RNase PH domain in hPNPase. (E) Trimeric and monomeric full-length structural models of hPNPase generated by superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K) with those of S. antibioticus PNPase (PDB entry 1E3P) and S1 domain of E. coli PNPase (PDB entry 1SRO). Blue, magenta and yellow represent S1 domain, KH domain and RNase PH domain, respectively.

Mentions: Each monomeric subunit of ΔS1 hPNPase in the crystal structure contained two visible RNase PH domains, one α-helical domain, one C-terminal KH domain and two citrate ions originating from the crystallization buffer (Figure 2A). In the trimeric ΔS1 hPNPase, the six RNase PH domains assembled into a ring-like structure with a central channel for RNA binding and cleavage (Figure 2B). The two citrates were bound in the RNA-binding channel near the active site in the second RNase PH domain of hPNPase, which displayed a geometry similar to those found in E. coli PNPase (25). The residues Arg446, Ser484, Asp538 and Asp544, which are critical for the phosphorylase activity of hPNPase as identified by previous site-directed mutagenesis studies (34), were located closely to the bound citrates, thus suggesting that the citrate ions were bound at the active site (see Figure 2D).Figure 2.


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)

Crystal structure of ΔS1 hPNPase. (A) The monomeric structure of ΔS1 hPNPase comprises two RNase PH domains (blue and yellow), an α-helical domain (green) and a KH domain (magenta). Two citrate ions (orange) bound near the active site are displayed in stick models. (B) Side view and top view of the trimeric ΔS1 hPNPase which assembled into a ring-like structure with a central channel. The three KH domains are located on the top of the ring, forming a novel RNA-binding KH pore. (C) Superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K, magenta) with those of S. antibioticus PNPase (PDB entry 1E3P, yellow) and E. coli PNPase (PDB entry 3CDI, gray). The RNase PH and α-helical domains matched well; however, the KH domain in hPNPase (marked by a dashed black oval) does not superimpose with the poly-alanine chain in S. antibioticus PNPase (marked by a dashed red oval). (D) Two citrate ions (stick models in orange) bind at the active site in the second RNase PH domain in hPNPase. (E) Trimeric and monomeric full-length structural models of hPNPase generated by superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K) with those of S. antibioticus PNPase (PDB entry 1E3P) and S1 domain of E. coli PNPase (PDB entry 1SRO). Blue, magenta and yellow represent S1 domain, KH domain and RNase PH domain, respectively.
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Related In: Results  -  Collection

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gkr1281-F2: Crystal structure of ΔS1 hPNPase. (A) The monomeric structure of ΔS1 hPNPase comprises two RNase PH domains (blue and yellow), an α-helical domain (green) and a KH domain (magenta). Two citrate ions (orange) bound near the active site are displayed in stick models. (B) Side view and top view of the trimeric ΔS1 hPNPase which assembled into a ring-like structure with a central channel. The three KH domains are located on the top of the ring, forming a novel RNA-binding KH pore. (C) Superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K, magenta) with those of S. antibioticus PNPase (PDB entry 1E3P, yellow) and E. coli PNPase (PDB entry 3CDI, gray). The RNase PH and α-helical domains matched well; however, the KH domain in hPNPase (marked by a dashed black oval) does not superimpose with the poly-alanine chain in S. antibioticus PNPase (marked by a dashed red oval). (D) Two citrate ions (stick models in orange) bind at the active site in the second RNase PH domain in hPNPase. (E) Trimeric and monomeric full-length structural models of hPNPase generated by superimposition of the structure of ΔS1 hPNPase (PDB entry 3U1K) with those of S. antibioticus PNPase (PDB entry 1E3P) and S1 domain of E. coli PNPase (PDB entry 1SRO). Blue, magenta and yellow represent S1 domain, KH domain and RNase PH domain, respectively.
Mentions: Each monomeric subunit of ΔS1 hPNPase in the crystal structure contained two visible RNase PH domains, one α-helical domain, one C-terminal KH domain and two citrate ions originating from the crystallization buffer (Figure 2A). In the trimeric ΔS1 hPNPase, the six RNase PH domains assembled into a ring-like structure with a central channel for RNA binding and cleavage (Figure 2B). The two citrates were bound in the RNA-binding channel near the active site in the second RNase PH domain of hPNPase, which displayed a geometry similar to those found in E. coli PNPase (25). The residues Arg446, Ser484, Asp538 and Asp544, which are critical for the phosphorylase activity of hPNPase as identified by previous site-directed mutagenesis studies (34), were located closely to the bound citrates, thus suggesting that the citrate ions were bound at the active site (see Figure 2D).Figure 2.

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