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The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

Kljashtorny V, Nikonov S, Ovchinnikov L, Lyabin D, Vodovar N, Curmi P, Manivet P - PLoS ONE (2015)

Bottom Line: Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA.The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity.The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia; Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Bd François Mitterrand, 91025 Evry Cedex, France; Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS 942, Hôpital Lariboisière, 41 boulevard de la Chapelle, 75475 Paris cedex 10, France; Assistance Publique-Hôpitaux de paris (APHP), Hôpital Lariboisière, Service de Biochimie et de Biologie Moléculaire, Paris, France.

ABSTRACT
The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

No MeSH data available.


Related in: MedlinePlus

Determination of the apparent dissociation constants (Ka) of the complexes of recombinant YB-1 (A,B) and CSD (C,D) with RNA and DNA.A and C, Chemically synthesized RNA or DNA fragments of similar sequence and length (7 pmol) were incubated with increasing amounts of recombinant YB-1 (A) or CSD (C) in 10 μl of reaction buffer. The resultant RNA-protein or DNA-protein complexes were analyzed by 8% PAGE followed by silver staining. Protein/NA molar ratio are mentioned at the top of the figures for each experimental condition. B and D, Binding curves were drawn according to quantification of band intensities for free RNA or DNA using ImageJ software. The apparent dissociation constant (Ka) was estimated as protein concentration sufficient for 50% saturation. Solid and dotted lines show the RNA-protein and DNA-protein complex, respectively.
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pone.0130318.g007: Determination of the apparent dissociation constants (Ka) of the complexes of recombinant YB-1 (A,B) and CSD (C,D) with RNA and DNA.A and C, Chemically synthesized RNA or DNA fragments of similar sequence and length (7 pmol) were incubated with increasing amounts of recombinant YB-1 (A) or CSD (C) in 10 μl of reaction buffer. The resultant RNA-protein or DNA-protein complexes were analyzed by 8% PAGE followed by silver staining. Protein/NA molar ratio are mentioned at the top of the figures for each experimental condition. B and D, Binding curves were drawn according to quantification of band intensities for free RNA or DNA using ImageJ software. The apparent dissociation constant (Ka) was estimated as protein concentration sufficient for 50% saturation. Solid and dotted lines show the RNA-protein and DNA-protein complex, respectively.

Mentions: To verify this hypothesis, we evaluated the affinity of CSDYB-1 to G-rich DNA (5’-AGGATGGGTGAGTGAGGTAG-3’) and RNA (5’-AGGAUGGGUGAGUGAGGUAG-3’) oligonucleotides in vitro. Both oligonucleotides were incubated with increasing amount of recombinant full-length YB-1 or CSDYB-1. Quantification of the band intensity of each complex allowed estimating the apparent dissociation constants (Fig 7 and S7 Fig). Both YB-1 (Fig 7A and 7B) and CSDYB-1 (Fig 7C and 7D) bind RNA with more affinity than DNA: KD was 0.3 μM and 0.6 μM for the YB-1:RNA and the YB-1:DNA complexes, respectively. KD of CSDYB-1 complexes with RNA and DNA were one order of magnitude lower: 2.5 μM and 6 μM, respectively. These results are in line with our predictions from MDS.


The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

Kljashtorny V, Nikonov S, Ovchinnikov L, Lyabin D, Vodovar N, Curmi P, Manivet P - PLoS ONE (2015)

Determination of the apparent dissociation constants (Ka) of the complexes of recombinant YB-1 (A,B) and CSD (C,D) with RNA and DNA.A and C, Chemically synthesized RNA or DNA fragments of similar sequence and length (7 pmol) were incubated with increasing amounts of recombinant YB-1 (A) or CSD (C) in 10 μl of reaction buffer. The resultant RNA-protein or DNA-protein complexes were analyzed by 8% PAGE followed by silver staining. Protein/NA molar ratio are mentioned at the top of the figures for each experimental condition. B and D, Binding curves were drawn according to quantification of band intensities for free RNA or DNA using ImageJ software. The apparent dissociation constant (Ka) was estimated as protein concentration sufficient for 50% saturation. Solid and dotted lines show the RNA-protein and DNA-protein complex, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493011&req=5

pone.0130318.g007: Determination of the apparent dissociation constants (Ka) of the complexes of recombinant YB-1 (A,B) and CSD (C,D) with RNA and DNA.A and C, Chemically synthesized RNA or DNA fragments of similar sequence and length (7 pmol) were incubated with increasing amounts of recombinant YB-1 (A) or CSD (C) in 10 μl of reaction buffer. The resultant RNA-protein or DNA-protein complexes were analyzed by 8% PAGE followed by silver staining. Protein/NA molar ratio are mentioned at the top of the figures for each experimental condition. B and D, Binding curves were drawn according to quantification of band intensities for free RNA or DNA using ImageJ software. The apparent dissociation constant (Ka) was estimated as protein concentration sufficient for 50% saturation. Solid and dotted lines show the RNA-protein and DNA-protein complex, respectively.
Mentions: To verify this hypothesis, we evaluated the affinity of CSDYB-1 to G-rich DNA (5’-AGGATGGGTGAGTGAGGTAG-3’) and RNA (5’-AGGAUGGGUGAGUGAGGUAG-3’) oligonucleotides in vitro. Both oligonucleotides were incubated with increasing amount of recombinant full-length YB-1 or CSDYB-1. Quantification of the band intensity of each complex allowed estimating the apparent dissociation constants (Fig 7 and S7 Fig). Both YB-1 (Fig 7A and 7B) and CSDYB-1 (Fig 7C and 7D) bind RNA with more affinity than DNA: KD was 0.3 μM and 0.6 μM for the YB-1:RNA and the YB-1:DNA complexes, respectively. KD of CSDYB-1 complexes with RNA and DNA were one order of magnitude lower: 2.5 μM and 6 μM, respectively. These results are in line with our predictions from MDS.

Bottom Line: Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA.The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity.The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia; Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Bd François Mitterrand, 91025 Evry Cedex, France; Institut National de la Santé et de la Recherche Médicale (INSERM), UMRS 942, Hôpital Lariboisière, 41 boulevard de la Chapelle, 75475 Paris cedex 10, France; Assistance Publique-Hôpitaux de paris (APHP), Hôpital Lariboisière, Service de Biochimie et de Biologie Moléculaire, Paris, France.

ABSTRACT
The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD). Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP), albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

No MeSH data available.


Related in: MedlinePlus