An Evolved RNA Recognition Motif That Suppresses HIV‑1 Tat/TAR-Dependent Transcription

Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents–small molecule, nucleic acid, or protein–reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognitionand modulationof disease-relevant RNA

Chromatin changes during AEC differentiation.

<p>A) Manhattan plot of differential chromatin changes. X-axis = chromosomal location, Y-axis = number of cell type-specific chromatin changes within 2 MB region. Upper panel = H3K9/14^Ac changes, blue = AT2 cell-specific acetylation, purple = AT1 cell-specific acetylation. Lower panel = H3K27^me3 changes, orange = AT2 cell-specific methylation, grey = AT1 cell-specific methylation. B) 135 TFBS enrichment in domains of chromatin change from HOMER. X-axis = H3K9/14^Ac, Y-axis = H3K27^me3 enrichment. AT2 enrichment is shown as the log₁₀ TFBS p-value, AT1 enrichment is shown as the −log₁₀ TFBS p-value. C) Example of chromatin changes at an upregulated gene, <i>FZD2</i>, using IGV to visualize chromatin tracks. Blue = H3K9/14^Ac raw reads and SICER peaks called, green = predicted RXR binding site from HOMER analysis. D) Example of downregulated gene expression at the <i>PGC</i> gene locus. Lavender = predicted FOXA1 binding sites from HOMER analysis. AT2 = AEC chromatin signature (D0), AT1 = AEC chromatin signature (D8).</p

Transcriptomic profiling of human AEC differentiation.

<p>A) Heatmap of top 5% variant-VSN normalized gene expression probes. Blue = low expression, red = high expression. DAY = number of days AT2 cells were allowed to differentiate. “Prep” = donor lung origin by color (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003513#pgen.1003513.s001" target="_blank">Figure S1</a>). B) Principal component analysis of normalized hAEC samples. Samples color coded by donor lung as in (A). C) Significant changes in hAEC gene expression. Black line = BH-adjusted cutoff (FDR adjusted p≤0.05) calculated between D0 and D8. 20 genes show both significant up and downregulation for probes in different locations of the gene. D) Manhattan plot of differentially expressed genes. X-axis = chromosomal location, Y-axis = number of genes in each 2 MB region. E) qRT-PCR validation of microarray, data expressed in log₂-fold change of differences between D0 and D8. Circles = top 10 up- and down-regulated genes, triangles = known AT1 cell differentiation markers (<i>AQP5, PDPN, CAV1</i>). F) IPA of significantly up- or down-regulated genes. Bars expressed as log₁₀-BH corrected p-values of enrichment for pathway members in significant list against RefSeq db38 background. Whole figure: Red = upregulated, green = downregulated.</p

Integration of gene expression data with epigenetic alterations.

<p>A–B–C) Relationship between all 16 possible combinations of chromatin changes and gene expression. Grey = unassociated with H3K9/14^Ac or H3K27^me3 changes, red = potentially activating chromatin changes, green = potentially repressive chromatin changes, blue = mixture of both. A) Significant expression changes in genes as a percentage of all genes associated with each histone mark for each of the possible 16 combinations of chromatin marks. Upregulated = above x-axis, downregulated = below x-axis. B) Total number of genes with significant gene expression changes associated with each chromatin combination. C) Representative IPA network of upregulated genes with both H3K9/14^Ac gain and H3K27^me3 loss. D) Representative IPA network of downregulated genes with H3K9/14^Ac loss. E and F) IPA ranked networks of genes subset by chromatin context. Corresponding TFBS present in subset chromatin and enrichment p-value from HOMER analysis, for each chromatin-associated gene subset. Red = upregulated gene expression and activating chromatin changes, green = downregulated gene expression and deactivating chromatin changes. (*) Indicates below significance threshold in HOMER but still present in IPA.</p

Functional validation of a transcription factor signaling pathway predicted from bioinformatics analysis.

<p>A) Western blots examining AT2 and AT1 cell markers during differentiation in the presence or absence of RXR antagonist UVI-3003. LAMIN A/C is the loading control. B) Transepithelial resistance as measured in kΩ-cm² over the course of differentiation. Error bars represent technical duplicates for each plating. C) Rat <i>Aqp5</i>-luciferase 4.3 kb promoter construct. Grey lines = 34 putative PPARA:RXR binding sites (Explain3.0). No sites were predicted from −900 to +6 bp due to lack of rat sequence information in the Explain v3.0 database. The asterisk marks the approximate location in the promoter of the ChIPed RXR site in E, below. The average number of PPARA:RXR sites per kilobase in the listed human/rat/mouse promoters is given in the table, with consensus site listed at the top. D) MLE-15 cells were transiently transfected with the <i>Aqp5</i>-luciferase construct and treated for 48 hours with vehicle (DMSO) or 7.5 µM UVI-3003. UV1-3003 treatment reduced <i>Aqp5</i>-luc activity by 48%±0.06. Values were normalized to vehicle control and represent the mean, error bars represent SEM, N = 3. All experiments represent 3 biological replicates. E) ChIP was performed on primary cultured rat AEC at day 0 (AT2, D0, n = 2) and day 8 (AT1-like, D8, n = 3). A region ∼4 kb upstream of the transcription start site specifically precipitated with RXR in day 8 samples. ChIP of GAPDH with RXR was performed as a control, and POL2 (POLR2A) binding to the GAPDH promoter was included as a positive control for the quality of day 0 DNA.</p

Comparative transcriptomic profiling of human and rat AEC differentiation.

<p>A) Heatmap of top 2% of variant-VSN normalized gene expression probes in rat AEC. Blue = low expression, red = high expression. Prep = separate rAEC purifications. B) Significant changes in rAEC gene expression: red = upregulated, green = downregulated, black line = BH-adjusted cutoff for significance (FDR adjusted p≤0.05) calculated between D0 and D8. C) Correlation between rAEC and hAEC statistically significant genes. Data points expressed as significance of change between D0 and D8. Direction of change derived from increase or decrease in gene expression. Red = statically significant upregulated genes in both hAEC and rAEC, green = statistically significant downregulated genes in both hAEC and rAEC. Dotted lines = BH-adjusted cutoff for significance (p.adjusted≤0.05) calculated between D0 and D8. D) Venn diagram of statistically significant gene overlap between hAEC and rAEC (Top), genes upregulated between hAEC and rAEC (Middle), and genes downregulated between hAEC and rAEC (Bottom). 271 genes were significant in both species but expression changed in opposite directions. In all three diagrams: pale color = hAEC-specific statistically significant gene expression changes, medium color = statistically significant overlap in both rAEC and hAEC, dark color = rAEC-specific statistically significant gene expression changes.</p