PSD-95 MRNA Translation Regulation by the Fragile X Mental Retardation Protein

Abstract

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...