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Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha.

Weber J, Bao H, Hartlmüller C, Wang Z, Windhager A, Janowski R, Madl T, Jin P, Niessing D - Elife (2016)

Bottom Line: It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome.Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function.By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany.

ABSTRACT
The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

Drosophila Pur-alpha repeat I-II mutants show decreased binding affinity to DNA and RNA and decreased dsDNA-unwinding activity.(A–E) Radioactive EMSA with wild-type or mutant Pur-alpha repeat I-II and with MF0677 ssDNA (left) or RNA (middle) and CGG-repeat RNA (right). EMSA with CGG-repeat DNA is shown in Figure 3A–E. All mutants show decreased nucleic acid binding, except for the F68 mutant (F I) (D), the counterpart to F145 on repeat II (F II). Open arrowheads indicate free and filled arrowheads indicate protein-bound DNA/RNA oligonucleotides. (F) Mutations in repeat I (KNR I) (left) or in the identical motif in repeat II (KNR II) (middle) decreased the unwinding activity. (Right) Decreased unwinding also occurs upon mutation of F I.DOI:http://dx.doi.org/10.7554/eLife.11297.014
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fig3s1: Drosophila Pur-alpha repeat I-II mutants show decreased binding affinity to DNA and RNA and decreased dsDNA-unwinding activity.(A–E) Radioactive EMSA with wild-type or mutant Pur-alpha repeat I-II and with MF0677 ssDNA (left) or RNA (middle) and CGG-repeat RNA (right). EMSA with CGG-repeat DNA is shown in Figure 3A–E. All mutants show decreased nucleic acid binding, except for the F68 mutant (F I) (D), the counterpart to F145 on repeat II (F II). Open arrowheads indicate free and filled arrowheads indicate protein-bound DNA/RNA oligonucleotides. (F) Mutations in repeat I (KNR I) (left) or in the identical motif in repeat II (KNR II) (middle) decreased the unwinding activity. (Right) Decreased unwinding also occurs upon mutation of F I.DOI:http://dx.doi.org/10.7554/eLife.11297.014

Mentions: First, radioactive EMSA were performed with CGG-repeat and MF0677 DNA/RNA oligomers (24 nt). Except for Pur-alpha mutant F I, all other mutants showed decreased binding to DNA and RNA oligonucleotides with both motifs (Figure 3A–E, G; Figure 3—figure supplement 1A–E). In order to quantify these interactions, we performed fluorescence-polarization experiments with fluorescein-labeled MF0677 DNA and different variants of Pur-alpha. The effects observed in EMSA of mutations in Pur-alpha I-II were confirmed by these experiments (Figure 3H; Figure 3—figure supplement 2). Of note, mutations in PUR repeat I (KNR I, F I) had less severe effects on DNA binding than mutations in repeat II (KNR II, F II).10.7554/eLife.11297.013Figure 3.Mutations in Pur-alpha repeat I-II decrease nucleic-acid binding and dsDNA unwinding. 


Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha.

Weber J, Bao H, Hartlmüller C, Wang Z, Windhager A, Janowski R, Madl T, Jin P, Niessing D - Elife (2016)

Drosophila Pur-alpha repeat I-II mutants show decreased binding affinity to DNA and RNA and decreased dsDNA-unwinding activity.(A–E) Radioactive EMSA with wild-type or mutant Pur-alpha repeat I-II and with MF0677 ssDNA (left) or RNA (middle) and CGG-repeat RNA (right). EMSA with CGG-repeat DNA is shown in Figure 3A–E. All mutants show decreased nucleic acid binding, except for the F68 mutant (F I) (D), the counterpart to F145 on repeat II (F II). Open arrowheads indicate free and filled arrowheads indicate protein-bound DNA/RNA oligonucleotides. (F) Mutations in repeat I (KNR I) (left) or in the identical motif in repeat II (KNR II) (middle) decreased the unwinding activity. (Right) Decreased unwinding also occurs upon mutation of F I.DOI:http://dx.doi.org/10.7554/eLife.11297.014
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4764581&req=5

fig3s1: Drosophila Pur-alpha repeat I-II mutants show decreased binding affinity to DNA and RNA and decreased dsDNA-unwinding activity.(A–E) Radioactive EMSA with wild-type or mutant Pur-alpha repeat I-II and with MF0677 ssDNA (left) or RNA (middle) and CGG-repeat RNA (right). EMSA with CGG-repeat DNA is shown in Figure 3A–E. All mutants show decreased nucleic acid binding, except for the F68 mutant (F I) (D), the counterpart to F145 on repeat II (F II). Open arrowheads indicate free and filled arrowheads indicate protein-bound DNA/RNA oligonucleotides. (F) Mutations in repeat I (KNR I) (left) or in the identical motif in repeat II (KNR II) (middle) decreased the unwinding activity. (Right) Decreased unwinding also occurs upon mutation of F I.DOI:http://dx.doi.org/10.7554/eLife.11297.014
Mentions: First, radioactive EMSA were performed with CGG-repeat and MF0677 DNA/RNA oligomers (24 nt). Except for Pur-alpha mutant F I, all other mutants showed decreased binding to DNA and RNA oligonucleotides with both motifs (Figure 3A–E, G; Figure 3—figure supplement 1A–E). In order to quantify these interactions, we performed fluorescence-polarization experiments with fluorescein-labeled MF0677 DNA and different variants of Pur-alpha. The effects observed in EMSA of mutations in Pur-alpha I-II were confirmed by these experiments (Figure 3H; Figure 3—figure supplement 2). Of note, mutations in PUR repeat I (KNR I, F I) had less severe effects on DNA binding than mutations in repeat II (KNR II, F II).10.7554/eLife.11297.013Figure 3.Mutations in Pur-alpha repeat I-II decrease nucleic-acid binding and dsDNA unwinding. 

Bottom Line: It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome.Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function.By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München - German Research Center for Environmental Health, Neuherberg, Germany.

ABSTRACT
The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus