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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 RNA-binding central channel in PNPase and exosomes. (A) Comparison between the central channel in E. coli PNPase (ePNPase, PDB: 3CDI), S. antibioticus PNPase (sPNPase, PDB: 1E3P) and hPNPase (PDB: 3U1K) shows that hPNPase has the most constricted channel for RNA binding among these PNPases [calculated by using the program HOLE (47)]. The channel regions with a diameter of <5 Å are displayed in red and pink. The neck regions are marked by black arrows and the active sites are marked by red arrows. (B) The crystal structures of human exosome (PDB: 2NN6) and S. solfataricus exosome (PDB: 2JE6) show that the active S. solfataricus exosome has a more constricted channel as compared to the inactive human exosome. (C) Structural model of hPNPase bound with a structured RNA.
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gkr1281-F7: The RNA-binding central channel in PNPase and exosomes. (A) Comparison between the central channel in E. coli PNPase (ePNPase, PDB: 3CDI), S. antibioticus PNPase (sPNPase, PDB: 1E3P) and hPNPase (PDB: 3U1K) shows that hPNPase has the most constricted channel for RNA binding among these PNPases [calculated by using the program HOLE (47)]. The channel regions with a diameter of <5 Å are displayed in red and pink. The neck regions are marked by black arrows and the active sites are marked by red arrows. (B) The crystal structures of human exosome (PDB: 2NN6) and S. solfataricus exosome (PDB: 2JE6) show that the active S. solfataricus exosome has a more constricted channel as compared to the inactive human exosome. (C) Structural model of hPNPase bound with a structured RNA.

Mentions: In PNPase and exosomes, RNA is bound in the central channel of the ring-like structures. It is intriguing to ponder what kind of channel might be more efficient for RNA binding. A previous study suggests that the KH/S1 domains are not only involved in RNA binding but also in the formation of a compact trimer with a constricted central channel (24). For example, the Sulfolobus solfataricus exosome Rrp41/Rrp42 core complex has a wider central channel than the holoenzyme Rrp4/Rrp41/Rrp42. The central channel of KH/S1 domain-truncated E. coli PNPase is also larger than that of full-length protein. Comparing the central channel of three PNPase structures, we found that E. coli PNPase has the widest central channel, likely because the KH/S1 domains are not present, while S. antibioticus PNPase has a more constricted central channel because it has a partially ordered KH/S1 RNA-binding domain (Figure 7A). Remarkably, hPNPase has an even more constricted central channel with two neck regions, one within the KH domain and one in a region on top of the RNase PH ring (displayed in red and pink in Figure 7A). We also noticed that the inactive human exosome has a wide channel, whereas the active S. solfataricus exosome has a narrow channel with a constricted neck region (Figure 7B). This result suggests that the RNA-binding KH domain in PNPase regulates the size of the channel and therefore in turn regulates the RNA-binding activity as well as the RNA cleavage activity of PNPase. A more constricted central channel appears more efficient for RNA binding and cleavage.Figure 7.


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 RNA-binding central channel in PNPase and exosomes. (A) Comparison between the central channel in E. coli PNPase (ePNPase, PDB: 3CDI), S. antibioticus PNPase (sPNPase, PDB: 1E3P) and hPNPase (PDB: 3U1K) shows that hPNPase has the most constricted channel for RNA binding among these PNPases [calculated by using the program HOLE (47)]. The channel regions with a diameter of <5 Å are displayed in red and pink. The neck regions are marked by black arrows and the active sites are marked by red arrows. (B) The crystal structures of human exosome (PDB: 2NN6) and S. solfataricus exosome (PDB: 2JE6) show that the active S. solfataricus exosome has a more constricted channel as compared to the inactive human exosome. (C) Structural model of hPNPase bound with a structured RNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1281-F7: The RNA-binding central channel in PNPase and exosomes. (A) Comparison between the central channel in E. coli PNPase (ePNPase, PDB: 3CDI), S. antibioticus PNPase (sPNPase, PDB: 1E3P) and hPNPase (PDB: 3U1K) shows that hPNPase has the most constricted channel for RNA binding among these PNPases [calculated by using the program HOLE (47)]. The channel regions with a diameter of <5 Å are displayed in red and pink. The neck regions are marked by black arrows and the active sites are marked by red arrows. (B) The crystal structures of human exosome (PDB: 2NN6) and S. solfataricus exosome (PDB: 2JE6) show that the active S. solfataricus exosome has a more constricted channel as compared to the inactive human exosome. (C) Structural model of hPNPase bound with a structured RNA.
Mentions: In PNPase and exosomes, RNA is bound in the central channel of the ring-like structures. It is intriguing to ponder what kind of channel might be more efficient for RNA binding. A previous study suggests that the KH/S1 domains are not only involved in RNA binding but also in the formation of a compact trimer with a constricted central channel (24). For example, the Sulfolobus solfataricus exosome Rrp41/Rrp42 core complex has a wider central channel than the holoenzyme Rrp4/Rrp41/Rrp42. The central channel of KH/S1 domain-truncated E. coli PNPase is also larger than that of full-length protein. Comparing the central channel of three PNPase structures, we found that E. coli PNPase has the widest central channel, likely because the KH/S1 domains are not present, while S. antibioticus PNPase has a more constricted central channel because it has a partially ordered KH/S1 RNA-binding domain (Figure 7A). Remarkably, hPNPase has an even more constricted central channel with two neck regions, one within the KH domain and one in a region on top of the RNase PH ring (displayed in red and pink in Figure 7A). We also noticed that the inactive human exosome has a wide channel, whereas the active S. solfataricus exosome has a narrow channel with a constricted neck region (Figure 7B). This result suggests that the RNA-binding KH domain in PNPase regulates the size of the channel and therefore in turn regulates the RNA-binding activity as well as the RNA cleavage activity of PNPase. A more constricted central channel appears more efficient for RNA binding and cleavage.Figure 7.

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