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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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Structural model of RNA bound at the KH pore of hPNPase. (A) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the SF1–RNA complex structure (PDB entry 1K1G, beige for SF1 and orange for RNA) gave an average RMSD of 3.12 Å for 30 Cα atoms. (B) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the Nova2–RNA complex structure (PDB entry 1EC6, gray for Nova2 and orange for RNA) gave an average RMSD of 1.45 Å for 48 Cα atoms. (C) Structural model of the KH domain (magenta) of hPNPase in complex with a single-stranded, 4-nt RNA adapted from Nova2-RNA. The GXXG motif is marked by a dashed circle. (D) Structural model of the hPNPase–RNA complex. The 4-nt RNA is bound inside the KH pore. (E) A close-up view of the trimeric KH pore in hPNPase bound with RNA. The 3′-end of the RNA points inward into the channel.
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gkr1281-F4: Structural model of RNA bound at the KH pore of hPNPase. (A) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the SF1–RNA complex structure (PDB entry 1K1G, beige for SF1 and orange for RNA) gave an average RMSD of 3.12 Å for 30 Cα atoms. (B) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the Nova2–RNA complex structure (PDB entry 1EC6, gray for Nova2 and orange for RNA) gave an average RMSD of 1.45 Å for 48 Cα atoms. (C) Structural model of the KH domain (magenta) of hPNPase in complex with a single-stranded, 4-nt RNA adapted from Nova2-RNA. The GXXG motif is marked by a dashed circle. (D) Structural model of the hPNPase–RNA complex. The 4-nt RNA is bound inside the KH pore. (E) A close-up view of the trimeric KH pore in hPNPase bound with RNA. The 3′-end of the RNA points inward into the channel.

Mentions: The crystal structures of two proteins with a KH domain bound with RNA have been determined, neuro-oncological ventral antigen 2 (Nova2) KH3 (PDB entry 1EC6) and splicing factor 1 (SF1) KH (PDB entry 1K1G) (36,37). Superimposition of the KH domain of hPNPase with the Nova2 and SF1 KH–RNA complex structures gave an average RMSD of 1.45 Å for 48 Cα atoms and 3.12 Å for 30 Cα atoms, respectively (Figure 4A and B). This result suggests that the KH domain in hPNPase has a structure closely resembling those in Nova2 and SF1. Moreover, the RNA was bound in a similar manner in the KH domain on the edge of the α-helix and β-sheet in Nova2 and SF1. Therefore, a complex model of the hPNPase KH domain bound with an RNA molecule was built based on the structure of the Nova2–RNA complex. The hPNPase KH-RNA model had a 4-nt RNA bound in the RNA-binding cleft of the KH domain (Figure 4C). Superimposition of the bound RNA with the trimeric hPNPase further showed that the RNA substrate fitted right into the KH pore of the central channel. One of the three possible models is shown in Figure 4D and E. The sugar phosphate backbone of the 4-nt RNA in the KH pore was parallel to the channel direction, with the 3′-end pointing inward. This result implies that the RNA substrate threads through the KH pore to reach the central channel formed by the RNase PH domains for degradation.Figure 4.


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)

Structural model of RNA bound at the KH pore of hPNPase. (A) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the SF1–RNA complex structure (PDB entry 1K1G, beige for SF1 and orange for RNA) gave an average RMSD of 3.12 Å for 30 Cα atoms. (B) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the Nova2–RNA complex structure (PDB entry 1EC6, gray for Nova2 and orange for RNA) gave an average RMSD of 1.45 Å for 48 Cα atoms. (C) Structural model of the KH domain (magenta) of hPNPase in complex with a single-stranded, 4-nt RNA adapted from Nova2-RNA. The GXXG motif is marked by a dashed circle. (D) Structural model of the hPNPase–RNA complex. The 4-nt RNA is bound inside the KH pore. (E) A close-up view of the trimeric KH pore in hPNPase bound with RNA. The 3′-end of the RNA points inward into the channel.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1281-F4: Structural model of RNA bound at the KH pore of hPNPase. (A) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the SF1–RNA complex structure (PDB entry 1K1G, beige for SF1 and orange for RNA) gave an average RMSD of 3.12 Å for 30 Cα atoms. (B) Superimposition of the hPNPase KH domain (magenta) with the KH domain in the Nova2–RNA complex structure (PDB entry 1EC6, gray for Nova2 and orange for RNA) gave an average RMSD of 1.45 Å for 48 Cα atoms. (C) Structural model of the KH domain (magenta) of hPNPase in complex with a single-stranded, 4-nt RNA adapted from Nova2-RNA. The GXXG motif is marked by a dashed circle. (D) Structural model of the hPNPase–RNA complex. The 4-nt RNA is bound inside the KH pore. (E) A close-up view of the trimeric KH pore in hPNPase bound with RNA. The 3′-end of the RNA points inward into the channel.
Mentions: The crystal structures of two proteins with a KH domain bound with RNA have been determined, neuro-oncological ventral antigen 2 (Nova2) KH3 (PDB entry 1EC6) and splicing factor 1 (SF1) KH (PDB entry 1K1G) (36,37). Superimposition of the KH domain of hPNPase with the Nova2 and SF1 KH–RNA complex structures gave an average RMSD of 1.45 Å for 48 Cα atoms and 3.12 Å for 30 Cα atoms, respectively (Figure 4A and B). This result suggests that the KH domain in hPNPase has a structure closely resembling those in Nova2 and SF1. Moreover, the RNA was bound in a similar manner in the KH domain on the edge of the α-helix and β-sheet in Nova2 and SF1. Therefore, a complex model of the hPNPase KH domain bound with an RNA molecule was built based on the structure of the Nova2–RNA complex. The hPNPase KH-RNA model had a 4-nt RNA bound in the RNA-binding cleft of the KH domain (Figure 4C). Superimposition of the bound RNA with the trimeric hPNPase further showed that the RNA substrate fitted right into the KH pore of the central channel. One of the three possible models is shown in Figure 4D and E. The sugar phosphate backbone of the 4-nt RNA in the KH pore was parallel to the channel direction, with the 3′-end pointing inward. This result implies that the RNA substrate threads through the KH pore to reach the central channel formed by the RNase PH domains for degradation.Figure 4.

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