Multiple conserved structural cis-acting regulatory elements have been recognized both in the coding and untranslated regions (UTRs) of the hepatitis C virus (HCV) genome. For example, the cis-element 5BSL3.2 in the HCV-coding region has been predicted to use both its apical and internal loops to interact with the X RNA in the 3′-UTR, with the IIId domain in the 5′-UTR and with the Alt sequence in the coding region. Additionally, the X RNA region uses a palindromic sequence that overlaps the sequence required for the interaction with 5BSL3.2, to dimerize with another HCV genome. The ability of the 5BSL3.2 and X RNA regions to engage in multi-interactions suggests the existence of one or more molecular RNA switches which may regulate different steps of the HCV life cycle. In this study, we used biophysical methods to characterize the essential interactions of these HCV cis-elements at the molecular level. Our results indicate that X RNA interacts with 5BSL3.2 and another X RNA molecule by adopting two different conformations and that 5BSL3.2 engages simultaneously in kissing interactions using its apical and internal loops. Based on these results, we propose a mode of action for possible molecular switches involving the HCV RNA.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3 ′ -untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3 ′ UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site.

A novel posttranscriptional mechanism for regulating the neuronal protein GAP-43 is reported. The mRNA-binding protein hnRNP-Q1 represses Gap-43 mRNA translation by a mechanism involving a 5′ untranslated region G-quadruplex structure, which affects GAP-43 function, as demonstrated by a GAP-43–dependent increase in neurite length and number with hnRNP-Q1 knockdown.

Fragile X syndrome, the most common inherited form of intellectual disability, is caused by the CGG trinucleotide expansion in the 5’-untranslated region of the Fmr1 gene on the X chromosome, which silences the expression of the fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA, which encodes for the postsynaptic density protein 95, and together with microRNA-125a to mediate the reversible inhibition of the PSD-95 mRNA translation in neurons. The miR-125a binding site within the PSD-95 mRNA 3’-untranslated region (UTR) is embedded in a G-rich region bound by FMRP, which we have previously demonstrated folds into two parallel G-quadruplex structures. The FMRP regulation of PSD-95 mRNA translation is complex, being mediated by its phosphorylation. While the requirement for FMRP in the regulation of PSD-95 mRNA translation is clearly established, the exact mechanism by which this is achieved is not known. In this study, we have shown that both unphosphorylated FMRP and its phosphomimic FMRP S500D bind to the PSD-95 mRNA G-quadruplexes with high affinity, whereas only FMRP S500D binds to miR-125a. These results point towards a mechanism by which, depending on its phosphorylation status, FMRP acts as a switch that potentially controls the stability of the complex formed by the miR-125a-guided RNA induced silencing complex (RISC) and PSD-95 mRNA.

Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5′-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the Post Synaptic Density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3′-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3′-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-D-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets.

interpretation of ChIP results can be challenging when dealing with TFs exhibiting rapid turnover, or with cells and tissues exhibiting a patterned nonhomogeneous transcriptional response to an external stimulus. Here we describe a microscopy-based single molecule imaging approach which can be used to obtain direct information on the TF binding kinetics to chromatin with the sub-second temporal resolution at the individual live-cell level [1,2]. We apply this method to characterize the binding of the tumor suppressor p53 both in basal, non-stimulated conditions and upon its activation by genotoxic stress induced by ionizing radiation: we show that p53 binds transiently to DNA (timescale of seconds), and that this interaction is modulated following the induction of damage, Importantly, more stable interactions are associated to higher transcription rates of p53 target genes, indicating that p53 acts as a latent TF, reviving an hypothesis initially derived from in-vitro studies [3], but later challenged by low temporal resolution ChIP experiments [4].[1] D. Mazza et al., Nucl. More than 50% of cancer patients harbor p53 mutations, highlighting the essential role of p53 in tumor suppression. In response to various stress signals, p53 targets the TFIID-mediated transcription machinery to stimulate expression of vast gene networks involved in diverse cellular pathways. Understanding how p53 turns on TFIID-mediated transcription initiation at the single molecule level will advance our knowledge of p53's tumor suppression activity. Despite 20 years of biochemical studies, it remains elusive how p53 recruits TFIID and other basal transcription factors (e.g. TFIIB and RNA Polymerase II) to facilitate pre-initiation complex formation on various target gene promoters. We hypothesize that p53 induces distinct DNA-binding conformations within basal factors to stimulate assembly culminated in transcription initiation. Therefore, to test our hypothesis, we employed an innovative integrated approach involving single particle cryo-electron microscopy (EM), single molecule fluoresce microscopy and biochemistry. Intriguingly, our studies demonstrated that p53 delivers and promotes stable binding of TFIID to DNA. Unexpectedly, the interaction of TFIID and DNA in turn causes p53 to quickly dissociate from the assembly and promoter. Furthermore, our work suggest that the direct association of p53 and TFIID is sufficient to recruit TFIID onto various target gene promoters. More significantly, p53 induces a common DNA-binding conformation of TFIID. We further identified a novel role of TFIID in anchoring core promoter DNA downstream of the transcription start site. Collectively, these findings indicate that p53, and potentially activators in general, serve as escorts to dynamically recruit and load the basal transcription machinery onto DNA. Importantly, p53 facilitates formation of common DNA-binding forms of basal factors. Overall, our studies are critical for understanding how eukaryotic transcription complexes initiate transcription...

Historically, all small catalytic RNAs have been shown to undergo global conformational changes upon phosphodiester cleavage. However, the most recent of numerous hepatitis delta virus (HDV) ribozyme crystal structures has challenged this trend, as this crystal structure suggests that the precursor structure is already product-like in conformation. To further investigate this unusual observation, we have extensively characterized the solution behavior of several three-stranded versions of the HDV ribozyme from the recent crystal structure. Fluorescence gel shift assays show that varying lengths of the 5'overhang sequence adjacent to the active site result in the same degree of cleavage, whereas noncleavable substrates exhibit significantly more heterogeneity. Complementary steady state and time-resolved FRET assays demonstrated that the length of the 5'overhang sequence adjacent to the cleavage site affects the rates of conformational change upon substrate binding and cleavage. Molecular dynamics (MD) simulations were also performed to gain insight into the atomic behavior and catalytic relevance of the HDV ribozyme from the Chen et al crystal structure. These simulations suggested that the dU-1dG1dG2 motif used in the crystal does not result in a catalytically fit ribozyme compared to an all-ribose construct. Furthermore, altered active site conditions also result in lowered catalytic fitness. These simulations suggested that catalytic fitness is greatly disrupted by deprotonation of C75, supporting the hypothesis of the role of C75 as a general acid. Simulations also showed that the magnesium ion resolved near the scissile phosphate results in favorable catalytic geometry compared to simulations neutralized with sodium. Our experimental results demonstrate that, despite previously published results, all forms of the HDV ribozyme undergo significant global conformational changes upon self-cleavage, and our simulations show that C75 is poised to act as a general acid during cleavage. . microRNA-122 (miR-122), a liver specific microRNA, has been shown to facilitate the Hepatitis C virus (HCV) replication and/or translation. Although the exact role played by miR-122 in this process is not fully understood, it has been shown that one of the functions of miR-122 is to stabilize the HCV RNA genome upon binding. There are two miR-122 binding sites within the HCV genome 5'-untranslated region (5'-UTR), named the S1 and S2 sites, both containing the miR-122 seed sequence. It has been shown that miR-122 is a valid antiviral target, as locked nucleic acids developed against miR-122 abolished the HCV replication. However, miR-122 has numerous other functions in the hepatic cell, which will also be affected by these LNAs. In this study, we adopted a different approach, namely to design peptide nucleic acids (PNAs) against the miR-122 binding sites within the HCV genome, sites conserved in all HCV genotypes and to test their antiviral properties. Our results indicate that one such PNA designed against the site S2 binds to the...