Genome-wide analysis of heterogeneous nuclear ribonucleoprotein (hnRNP) binding to HIV-1 RNA reveals a key role for hnRNP H1 in alternative viral mRNA splicing

Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrore

Structural insights into calcium-bound S100P and the V domain of the RAGE complex.

The S100P protein is a member of the S100 family of calcium-binding proteins and possesses both intracellular and extracellular functions. Extracellular S100P binds to the cell surface receptor for advanced glycation end products (RAGE) and activates its downstream signaling cascade to meditate tumor growth, drug resistance and metastasis. Preventing the formation of this S100P-RAGE complex is an effective strategy to treat various disease conditions. Despite its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report that S100P preferentially binds to the V domain of RAGE. Furthermore, we characterized the interactions between the RAGE V domain and Ca(2+)-bound S100P using various biophysical techniques, including isothermal titration calorimetry (ITC), fluorescence spectroscopy, multidimensional NMR spectroscopy, functional assays and site-directed mutagenesis. The entropy-driven binding between the V domain of RAGE and Ca(+2)-bound S100P was found to lie in the micromolar range (Kd of ∼ 6 µM). NMR data-driven HADDOCK modeling revealed the putative sites that interact to yield a proposed heterotetrameric model of the S100P-RAGE V domain complex. Our study on the spatial structural information of the proposed protein-protein complex has pharmaceutical relevance and will significantly contribute toward drug development for the prevention of RAGE-related multifarious diseases

S100P-pentamidine interactions.

<p>(a) Overlaid ¹H-¹⁵N HSQC spectra of uniformly ¹⁵N-labeled free S100P (black) and S100P upon binding pentamidine at a 1∶1 molar ratio (red). (b) Thermodynamic studies of S100P with pentamidine as a ligand. Raw data (upper panels) and integrated heat measurements (lower panels) as a function of the molar ratio of the pentamidine to S100P protein. The dissociation constant (K_d) for this interaction is 305 µM at a 1∶1 stoichiometry. (c) Ribbon representation of the HADDOCK-modeled S100P-pentamidine complex. Monomers of the S100P homodimer are colored green and lemon green, with pentamidine shown as gray sticks. Structural elements of S100P are labeled in red. (d) Ribbon representation of the S100P-RAGE V domain complex with pentamidine at the binding interface. (e) Effects of pentamidine on S100P-mediated cell proliferation and RAGE signaling. SW-480 cells were treated with 10 µM FPS-ZM1, 100 µM S100P, 10 µM FPS-ZM1 plus 100 µM S100P or 10 µM pentamidine plus 100 µM S100P. Cell proliferation was analyzed after 48 h.</p

Analysis of the ¹H-¹⁵N HSQC spectra of the RAGE V domain in complex with S100P.

<p>(a) Overlaid ¹H-¹⁵N HSQC spectra of 0.3 mM free ¹⁵N-labeled RAGE V domain (black) and the RAGE V domain in complex with 0.3 mM unlabeled S100P domain (red). Spectral changes are indicated by blue boxes. The dashed line indicates the expansion spectra shown on the right. (b) Bar graph of the cross-peak intensity (I/I_o), where (I) is the cross-peak intensity of the RAGE V domain in complex with S100P and (I_o) is the initial intensity of the free RAGE V domain, versus the RAGE V domain residue number (residues 21-121). The residues are numbered as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103947#pone.0103947-Matsumoto1" target="_blank">[44]</a>. The black dashed line is the threshold of the selected residues that exhibited a significant decrease in intensity (<0.6). (c) Ribbon representation of the RAGE V domain, with the residues that exhibited a decrease in the cross-peak intensity was mapped (red color) in the ribbon diagram. β-strands and loops are labeled in black.</p

Functional assay.

<p>SW-480 cells were treated with 100 nM S100P, 100 nM S100P E5A, 100 nM S100P D13A, S100P F44G/Y88G/F89G or 10 µM FPS-ZM1, and cell proliferation was assessed using an MTT assay. The relative cell counts following treatment with the S100P mutants is plotted as the fold induction, with serum-free medium and FPS-ZM1 as the corresponding controls. The data are expressed as the means ± SD of 3 independent experiments.</p

Proposed mechanism for the S100P-RAGE interaction.

<p>A proposed mechanism for the calcium-bound S100P-RAGE signaling cascade. Ligation of extracellular RAGE by S100P promotes the homodimerization of the cytoplasmic domain of RAGE and results in the activation of two pathways, MAPK and NF-κB, leading to the downstream activation of ERK1/2 (extracellular signal-regulated kinase) and subsequent cellular proliferation.</p

HADDOCK structure calculation statistics of the 10 best S100P–RAGEV model structures.

<p>HADDOCK structure calculation statistics of the 10 best S100P–RAGEV model structures.</p

ITC titrations of the RAGE V domain with S100P mutants.

<p>(a) Ribbon representation of the model of the S100P-RAGE V domain complex with the mutated polar charged residues and hydrophobic residues of S100P shown as sticks. Helix-1, Helix-4 and loop-3 of S100P are colored cyan, and RAGE V is colored gray with the residues that interact with the mutated S100P residues shown as sticks. (b) Binding properties of S100P E5A, S100P D13A, and S100P F44G/Y88G/F89G with RAGE V domain. The upper panels represent the raw data and whereas the bottom panels are the integrated plot of the amount of heat liberated per injection as a function of the molar ratio of the S100P mutants to RAGE V domain. The dissociation constants (K_d) for the RAGE V domain-S100P E5A interaction and the RAGE V domain-S100P D13A interaction were determined to be 23.6 µM and 877.8 µM, respectively. No binding of S100P F44G/Y88G/F89G to the RAGE V domain was detected.</p

Model of the S100P-RAGE V domain complex determined by HADDOCK.

<p>(a) Backbone structures of an ensemble of the 10 lowest energy structures of the S100P-RAGE V domain complex. The S100P and RAGE V domain backbones are colored dark blue and red, respectively. (b) Ribbon and surface representation of the S100P-RAGE V domain complex, with the secondary structure elements of each RAGE V domain colored cyan and the secondary structure elements of the S100P homodimer colored green and lemon green.</p

Analysis of the ¹H-¹⁵N HSQC spectra of S100P in complex with the V domain of RAGE.

<p>(a) Overlaid ¹H-¹⁵N HSQC spectra of 0.3 mM ¹⁵N-labeled free S100P (black) and S100P in complex with 0.3 mM unlabeled RAGE V domain (red), with spectral changes indicated by blue boxes. The dashed line indicates the expansion spectra shown on the right. (b) Bar graph representing the changes in the cross-peak intensities (I/I_o) of free S100P and S100P in complex with the RAGE V domain versus the S100P residue number (1-95). In this plot, (I) represents the cross-peak intensity of S100P in complex with the RAGE V domain and (I_o) represents the initial intensity of free S100P. The black dashed line indicates the threshold of the selected residues that exhibited a significant decrease in the intensity (<0.6). (c) Ribbon representation of the S100P homodimer with the residues that exhibited a decrease in the cross-peak intensity was mapped (red color) in the ribbon diagram. The monomers of the S100P homodimer are colored green and lemon green.</p