The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling.

Analysis of mRNA abundance and stability by ribonuclease protection assay.

Gene expression is a multi-step process, which proceeds from DNA through RNA to protein. The tight regulation of this process is essential for overall cellular integrity and physiological homeostasis. Regulation of the messenger RNA (mRNA) levels has emerged as a crucial event in the modulation of the expression of genetic information.

The Nodal inhibitor Lefty is negatively modulated by the microRNA miR-302 in human embryonic stem cells.

MicroRNAs (miRNAs) have been shown to be important in early development and maintenance of human embryonic stem cells (hESCs). The miRNA miR-302-367 is specifically expressed in hESCs, and its expression decays on differentiation. We previously identified the structure of the gene coding for the human miR-302-367 cluster and characterized its promoter.

Inhibition of hepatitis C virus replication and internal ribosome entry site-dependent translation by an RNA molecule.

Hepatitis C virus (HCV) protein synthesis is mediated by a highly conserved internal ribosome entry site (IRES), mostly located at the 5' untranslatable region (UTR) of the viral genome. The translation mechanism is different from that used by cellular cap-mRNAs, making IRESs an attractive target site for new antiviral drugs. The present work characterizes a chimeric RNA molecule (HH363-50) composed of two inhibitors: a hammerhead ribozyme targeting position 363 of the HCV genome and an aptamer directed towards the essential stem-loop structure in domain IV of the IRES region (which contains the translation start codon).

The miR-302-367 cluster as a potential stemness regulator in ESCs.

Increasing experimental evidence suggests an important role of miRNAs in embryonic stem cell (ESC) biology. The miR-302-367 cluster is exclusively expressed at high levels in ESCs but not in either somatic stem cells or adult/embryonic differentiated cells. The human miR-302-367 gene structure has been recently described and its promoter has been identified, characterized and functionally validated in human stem cells.

Embryonic stem cell-specific miR302-367 cluster: human gene structure and functional characterization of its core promoter.

MicroRNAs (miRNAs) play a central role in the regulation of multiple biological processes including the maintenance of stem cell self-renewal and pluripotency. Recently, the miRNA cluster miR302-367 was shown to be differentially expressed in embryonic stem cells (ESCs). Unfortunately, very little is known about the genomic structure of miRNA-encoding genes and their transcriptional units.

Inhibition of HIV-1 replication by an improved hairpin ribozyme that includes an RNA decoy.

An anti-Tat hairpin ribozyme and a TAR RNA decoy were combined in one molecule. The chimeric molecule strongly inhibited HIV-1 replication (measured as changes in p24 levels in viral replication assays). The inhibitory action of the ribodecozyme (85%) was significantly greater than that shown by ribozyme and a non-catalytic variant carrying the functional decoy RNA domain (55% and 35%, respectively).

Inhibition of HIV-1 replication by RNA targeted against the LTR region.

Objective: The use of small RNA molecules able to effect gene inactivation has emerged as a powerful method of gene therapy. These small inhibitory RNAs are widely used for silencing malignant cellular and viral genes. We have assayed a series of inhibitory RNAs named catalytic antisense RNAs, consisting of a catalytic domain, hairpin or hammerhead ribozyme, and an antisense domain.

Interfering with hepatitis C virus IRES activity using RNA molecules identified by a novel in vitro selection method.

Hepatitis C virus (HCV) infection is one of the world's major health problems, and the identification of efficient HCV inhibitors is a major goal. Here we report the isolation of efficient anti-HCV internal ribosome entry site (IRES) RNA molecules identified by a new in vitro selection method. The newly developed procedure consists of two sequential steps that use distinct criteria for selection: selection for binding and selection for cleaving.

Design and optimization of sequence-specific hairpin ribozymes.

The hairpin ribozyme belongs to a group of small catalytic RNAs that have been extensively used to trans-cleave RNA molecules. Many efforts have been made to elucidate its reaction mechanism, and there is great interest in designing hairpin ribozymes with improved catalytic activity for use in the development of agents that specifically inactivate RNA molecules. This chapter summarizes the general principles in the design of hairpin ribozymes for targeting purposes, and provides a brief overview of the well-characterized modifications of the ribozyme sequences and structural domains that are necessary for optimal activity.

An experimental method for selecting effective target sites and designing hairpin ribozymes.

The proper selection of target sites and the correct design of specific ribozymes are decisive initial steps in any attempt to perform ribozyme-mediated gene silencing. Combinatorial methodologies can be used to improve ribozyme targeting and design. The in vitro selection strategy described in this chapter uses a combinatorial library of potentially self-cleaving RNA molecules.

HIV-1 TAR as anchoring site for optimized catalytic RNAs.

Ribozymes have a great potential for developing specific gene silencing molecules. One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target. Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence.

Ribozymes: recent advances in the development of RNA tools.

The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure.

Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions.

Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs.