Study of E. coli Hfq's RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents

Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other

The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation

The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44–61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing—both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44–61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal

YY1-binding sites provide central switch functions in the PARP-1 gene expression network.

Evidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (-574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (-587/-558), BM4 (-348/-319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis

The mouse minimal PARP-1 gene promoter, its binding motifs and extensions.

<p>(A) The <i>muPARP-1</i> core promoter as predicted by <i>Genomatix</i> (−572/+202 bp) as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044125#pone.0044125-Vidakovi1" target="_blank">[31]</a>. The 30 bp long oligonucleotides (BM 1 to 7) contain potential YY1 binding motifs and a negative control (BM6). Fragments 1 to 4 cover the entire promoter range with some overlaps (evaluated in Fig. 4). TSS – transcription start site (position +1); CDS – coding sequence. (B) Localization of YY1 biding motifs (BM1-7). The representation covers PARP-1 promoter upstream extension containing the functional PARP-1 binding motifs AGGCC (I), (highlighted in yellow and labelled with Roman numerals). The examined consensus PARP-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044125#pone.0044125-Vidakovi1" target="_blank">[31]</a> or YY1 sequences (in this paper) are framed by the red rectangles.</p

YY1 binds the Kozak sequence as the most prominent binding motif and assists PARP-1 binding.

<p>(A) Mutation of the YY1 core sequence within BM7 abolished YY1 binding as shown by super shift experiments with anti-YY1 antibody. (B) EMSA experiments performed with anti-PARP-1 antibody and selected mutated oligonucleotides m3 and m5 revealed that YY1 protein is required for PARP-1 binding to its consensus sequence located next to the Kozak sequence. The sequences of the double stranded oligonucleotides used as probes are as follows (small letters indicate the mutated positions): wildtype (BM7) 5′ ACG AGA AGG <b>A</b>GG <b><u>ATG</u><u> G</u></b>CG GAG GCC TCG GAG 3′ mutation 1 (m1) 5′ ACG Atc ctt <b>A</b>GG <b><u>ATG</u><u> G</u></b>CG GAG GCC TCG GAG 3′ mutation 2 (m2) 5′ ACG AGA AGt ctt cTG GCG GAG GCC TCG GAG 3′ mutation 3 (m3) 5′ ACG AGA AGG AGG cgt taG GAG GCC TCG GAG 3′ mutation 4 (m4) 5′ ACG AGA AGG <b>A</b>GG <b><u>ATG</u><u> G</u></b>at tct GCC TCG GAG 3′ mutation 5 (m5) 5′ ACG AGA AGG <b>A</b>GG <b><u>ATG</u><u> G</u></b>CG GAt taa gaG GAG 3′. Each probe (referred to as m1 to m5) was incubated in the absence or the presence of nuclear extract and examined by EMSA. Wild type BM7 was also incubated with nuclear extract and antibody to identify the bands that are shifted by YY1 or PARP-1 binding. Samples were run on a 8% polyacrylamide gel. The Kozak consensus sequence (gcc)gccRccATGG for which R is a purine three bases upstream of the start codon (AUG), is followed by another ‘G’, and is in bold capital letters.</p

YY1 binding affinity for the <i>muPARP-1</i> core promoter.

<p>(A) EMSA was performed with either the linearized or a circular, supercoiled 774 bp PARP-1 minimal promoter segment as part of the pSLGTKneo vector backbone. The assay involves incubation with recombinant PARP-1 protein or YY1 protein alone, or with both proteins at a 1∶1 molar ratio. Analyses are performed on non-denaturating 1% agarose gels. Complex formation for the linearized <i>muPARP-1</i> promoter fragment (“LIN”) and the vector-containing PARP-1 promoter (“SC”), was visualized with ethidium bromide. (B) The <i>in vivo</i> binding affinity of YY1 towards the PARP-1 promoter was confirmed by ChIP analysis with anti-YY1 antibody (H-414, Santa Cruz) as indicated. PARP-1 binding served as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044125#pone.0044125-Vidakovi1" target="_blank">[31]</a>. The anti-PARP-1 antibody was C2-10 from Alexis. Lane B – blank; no DNA template; lane 1– input DNA; 2– RNA pol II, positive control antibody; 3– IgG, negative control antibody; lane 4– NIH3T3 cell chromatin pull-down with YY1 antibody; 5– PARP^−/− cell chromatin pull-down with YY1 antibody; 6– NIH3T3 cell chromatin pull-down with PARP-1 antibody; 7– PARP^−/− cell chromatin pull-down with PARP-1 antibody.</p

Essential <i>muPARP-1</i> promoter regions identified in reporter plasmids.

<p>Reporter plasmids were pPARPlucTkneo, pPARPluc, pPARPluc<b>BM1^mut</b> (mutated YY1-binding motif in binding motif BM1) and pPARPluc<b>BM4^mut</b> (mutated YY1-binding motif in BM4). The YY1-binding motifs BM1, BM4 and BM7, the reporter gene translation start codon (ATG), the PARP-1 translation start codon (ATG*) and the stop codons that follow the PARP-1 gene translation start are indicated. The <i>muPARP-1</i> core-promoter predicted by <i>Genomatix</i> is contained in pPARPlucTkneo. To provide expression levels sufficient for the evaluation of PARP-1 promoter functions, the sequence must be extended upstream, but it has to exclude the translation start codon, the overlapping YY1-binding motif in BM7 and a minor part of the PARP-1 coding sequence. These changes permit analyses based on the luciferase (luc-) reporter as demonstrated in the inset. The corresponding analyses on mutants m1′ (ACATGG → cacgtG) and m2′ (CAATGT → CAcgtg) are applied to confirm increase of <i>muPARP-1</i> promoter activity relative to the wt sequences (Fig. 8).</p