Our view of RNA function changed a great deal from the discovery of messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), the deciphering of the genetic code, and of the roles these RNAs play in translation, during the 1950-1965 gold years. The 1960s-1980s period shone the spotlight on mRNA processing events and small RNAs that are part of small RNA-protein complexes essential for splicing. The discovery of RNA catalytic activity in the early 1980s transformed our thinking about RNA function, and this was just the beginning, as the discovery of both short and long noncoding RNAs in the 1990s and after 2000 triggered a new revolution in the RNA field. The microRNA (miRNA) story started with the discoveries in the Ambros and Ruvkun labs in the late 1980s-early 1990s that in C. elegans, lin-4, a repressor of the protein encoding lin-14, did not contain a conventional open reading frame, hence not encoding for a normal regulatory protein, but rather for a small 22 nucleotide noncoding RNA partially complementary in sequence to a stretch in the 3 ′-UTR of lin-14. This mechanism of gene expression regulation by a small RNA was unprecedented, but its generality was not clearly inferred, as research in the next decade did not bring an abundance of evidence of similar pairs of small noncoding RNAs that regulate protein expression through complementarity with 3 ′-UTR sequences in their mRNA targets. The discovery by Fire, Mello, and co-workers of RNA interference (RNAi), a process that involved small RNAs with a similar length to lin-4, hinted that this RNA too could have been generated by the same mechanisms operating in RNAi, and moreover, that other small noncoding RNAs that regulate gene expression might exist in C. elegans. This prediction was validated by the findings in the Ruvkun lab in 2000 of a second 21 nucleotide RNA, let-7, that controlled the expression of lin-41 by partial complementarity with a stretch of nucleotide in its 3 ′ UTR. Unlike the small noncoding RNAs involved in RNAi, that Hamilton and Baulcombe discovered had perfect complementarity to their targets leading to target destruction, lin-4 and let-7 had only partial complementarity to their mRNA targets

to O1, O2, and O3 operators in the lac operon. Specifically, we demonstrated that LacI kept two supercoils within the 401 bp DNA-loop between O1 and O2 operators. Additionally, We carried out time course studies to determine the stability of the topological barriers that are produced by the different LacI-operator complexes. Our results showed that the stability of the topological barriers correlates with the DNA-binding affinity of LacI to the different operators i.e., O1, O2, O3, and Os operators. Furthermore, we confirmed our previous observation in which LacI is able to ''keep'' certain superhelical energy to stabilize LacI-lacO1 complexes. Our results can be explained by a model in which LacI behaves as a topological barrier in the lac operon to regulate the expression of lac-ZYA genes in Escherichia coli cells.

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...

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation affecting approximately 1 in 4,000 males and 1 in 8,000 females. FXS is linked to the expansion of cytosine-guanine-guanine trinucleotide repeats in the fragile X mental retardation 1 (fmr1) gene. This expansion causes hypermethylation of the cytosines, transcriptional silencing of fmr1, and loss

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.

Fragile X syndrome, the most common form of inherited mental retardation in humans, affects about 1 in 3000 males and 1 in 5000 females. It is caused by the loss of expression of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5'-untranslated region (UTR) of the fragile x mental retardation-1 (fmr1) gene. FMRP has been shown to use its arginine-glycine-glycine (RGG) box RNA binding domain to bind with high affinity and specificity to G quadruplex forming mRNA sequences. The binding of FMRP to a proposed G quadruplex structure in the coding region of its own mRNA (100 nucleotide fragment named FBS) has been proposed to affect mRNA splicing events for isoforms 1 through 3. In this study we truncated the original 100 nt FMRP-FBS to 42 nt and used biophysical methods to directly demonstrate its folding into a G-quadruplex structure and the binding affinity of the different FMRP isoforms to it.