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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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RNA binding and cleavage assays for full-length (FL) and ΔS1 hPNPase. (A) EMSA assay showing that the RNA substrate binds with a similar affinity to full-length and ΔS1 hPNPase. The 12-mer poly(A) and poly(U) ssRNA substrate (0.1 pmol) was incubated, respectively, with hPNPase at various concentrations ranging from 1 to 16 µM in the absence of phosphate and Mg2+ ions. (B) The RNase activity of full-length and ΔS1 hPNPase was examined by incubation of the ssRNA with different concentrations of protein (0.1–0.8 µM) for 30 min at 37°C. Full-length and ΔS1 hPNPase exhibited a similar RNase activity. (C) Full-length and ΔS1 hPNPase can digest the long 3′ overhang (15 and 20 nt) of stem–loop RNA and generate major products with an overhang of 11–14 nt. The stem–loop RNA with a short 3′ overhang (7 and 10 nt) were more resistant for digestion. The marker (M) of 20-nt RNA corresponds to the stem–loop region of RNA with an 8-bp duplex and a 4-nt loop. The A0, A10 and A15 indicate the overhang size of 0, 10 and 15 nt, respectively.
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gkr1281-F5: RNA binding and cleavage assays for full-length (FL) and ΔS1 hPNPase. (A) EMSA assay showing that the RNA substrate binds with a similar affinity to full-length and ΔS1 hPNPase. The 12-mer poly(A) and poly(U) ssRNA substrate (0.1 pmol) was incubated, respectively, with hPNPase at various concentrations ranging from 1 to 16 µM in the absence of phosphate and Mg2+ ions. (B) The RNase activity of full-length and ΔS1 hPNPase was examined by incubation of the ssRNA with different concentrations of protein (0.1–0.8 µM) for 30 min at 37°C. Full-length and ΔS1 hPNPase exhibited a similar RNase activity. (C) Full-length and ΔS1 hPNPase can digest the long 3′ overhang (15 and 20 nt) of stem–loop RNA and generate major products with an overhang of 11–14 nt. The stem–loop RNA with a short 3′ overhang (7 and 10 nt) were more resistant for digestion. The marker (M) of 20-nt RNA corresponds to the stem–loop region of RNA with an 8-bp duplex and a 4-nt loop. The A0, A10 and A15 indicate the overhang size of 0, 10 and 15 nt, respectively.

Mentions: Previous structural analysis showed that the S1 domain in exosomes is involved in RNA binding. However, our crystal structure analysis suggests that the KH domain likely plays a dominant role for RNA binding in hPNPase through formation of a KH pore. To determine if the S1 domain in hPNPase is involved in RNA binding, full-length and ΔS1 hPNPase were incubated with poly(A)12 and poly(U)12 RNA in the absence of phosphate and magnesium ions. The electrophoretic mobility shift assay showed that the full-length and ΔS1 hPNPase bound poly(A)12 and poly(U)12 RNA with similar affinities. Moreover, ΔS1 hPNPase bound RNA almost as tightly as full-length hPNPase, thus suggesting that the S1 domain is not of critical importance for RNA binding (Figure 5A).Figure 5.


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)

RNA binding and cleavage assays for full-length (FL) and ΔS1 hPNPase. (A) EMSA assay showing that the RNA substrate binds with a similar affinity to full-length and ΔS1 hPNPase. The 12-mer poly(A) and poly(U) ssRNA substrate (0.1 pmol) was incubated, respectively, with hPNPase at various concentrations ranging from 1 to 16 µM in the absence of phosphate and Mg2+ ions. (B) The RNase activity of full-length and ΔS1 hPNPase was examined by incubation of the ssRNA with different concentrations of protein (0.1–0.8 µM) for 30 min at 37°C. Full-length and ΔS1 hPNPase exhibited a similar RNase activity. (C) Full-length and ΔS1 hPNPase can digest the long 3′ overhang (15 and 20 nt) of stem–loop RNA and generate major products with an overhang of 11–14 nt. The stem–loop RNA with a short 3′ overhang (7 and 10 nt) were more resistant for digestion. The marker (M) of 20-nt RNA corresponds to the stem–loop region of RNA with an 8-bp duplex and a 4-nt loop. The A0, A10 and A15 indicate the overhang size of 0, 10 and 15 nt, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351181&req=5

gkr1281-F5: RNA binding and cleavage assays for full-length (FL) and ΔS1 hPNPase. (A) EMSA assay showing that the RNA substrate binds with a similar affinity to full-length and ΔS1 hPNPase. The 12-mer poly(A) and poly(U) ssRNA substrate (0.1 pmol) was incubated, respectively, with hPNPase at various concentrations ranging from 1 to 16 µM in the absence of phosphate and Mg2+ ions. (B) The RNase activity of full-length and ΔS1 hPNPase was examined by incubation of the ssRNA with different concentrations of protein (0.1–0.8 µM) for 30 min at 37°C. Full-length and ΔS1 hPNPase exhibited a similar RNase activity. (C) Full-length and ΔS1 hPNPase can digest the long 3′ overhang (15 and 20 nt) of stem–loop RNA and generate major products with an overhang of 11–14 nt. The stem–loop RNA with a short 3′ overhang (7 and 10 nt) were more resistant for digestion. The marker (M) of 20-nt RNA corresponds to the stem–loop region of RNA with an 8-bp duplex and a 4-nt loop. The A0, A10 and A15 indicate the overhang size of 0, 10 and 15 nt, respectively.
Mentions: Previous structural analysis showed that the S1 domain in exosomes is involved in RNA binding. However, our crystal structure analysis suggests that the KH domain likely plays a dominant role for RNA binding in hPNPase through formation of a KH pore. To determine if the S1 domain in hPNPase is involved in RNA binding, full-length and ΔS1 hPNPase were incubated with poly(A)12 and poly(U)12 RNA in the absence of phosphate and magnesium ions. The electrophoretic mobility shift assay showed that the full-length and ΔS1 hPNPase bound poly(A)12 and poly(U)12 RNA with similar affinities. Moreover, ΔS1 hPNPase bound RNA almost as tightly as full-length hPNPase, thus suggesting that the S1 domain is not of critical importance for RNA binding (Figure 5A).Figure 5.

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