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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

Fluorescence-polarization measurements with wild type or various mutants of Pur-alpha I-II and MF0677 ssDNA.All experiments were performed as triplicates. KD values and standard deviations are given as insets in the plots. Curve fitting was performed as one-site binding.DOI:http://dx.doi.org/10.7554/eLife.11297.015
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fig3s2: Fluorescence-polarization measurements with wild type or various mutants of Pur-alpha I-II and MF0677 ssDNA.All experiments were performed as triplicates. KD values and standard deviations are given as insets in the plots. Curve fitting was performed as one-site binding.DOI:http://dx.doi.org/10.7554/eLife.11297.015

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)

Fluorescence-polarization measurements with wild type or various mutants of Pur-alpha I-II and MF0677 ssDNA.All experiments were performed as triplicates. KD values and standard deviations are given as insets in the plots. Curve fitting was performed as one-site binding.DOI:http://dx.doi.org/10.7554/eLife.11297.015
© Copyright Policy
Related In: Results  -  Collection

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

fig3s2: Fluorescence-polarization measurements with wild type or various mutants of Pur-alpha I-II and MF0677 ssDNA.All experiments were performed as triplicates. KD values and standard deviations are given as insets in the plots. Curve fitting was performed as one-site binding.DOI:http://dx.doi.org/10.7554/eLife.11297.015
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