Alfredo Berzal-Herranz

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme‐substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base‐pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second‐site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild‐type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme‐substrate complex are derived.

Embryonic Stem Cell-Specific miR302-367 Cluster: Human Gene Structure and Functional Characterization of Its Core Promoter

ABSTRACT 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. Here, we have characterized the structure of the gene coding for the human miR302-367 cluster. We identify the transcriptional start and functional core promoter region which specifically drives the expression of this miRNA cluster. The promoter activity depends on the ontogeny and hierarchical cellular stage. It is functional during embryonic development, but it is turned off later in development. From a hierarchical standpoint, its activity decays upon differentiation of ESCs, suggesting that its activity is restricted to the ESC compartment and that the ESC-specific expression of the miR302-367 cluster is fully conferred by its core promoter transcriptional activity. Furthermore, algorithmic prediction of transcription factor binding sites and knockdown studies suggest that ESC-associated transcription factors, including Nanog, Oct3/4, Sox2, and Rex1 may be upstream regulators of miR302-367 promoter. This study represents the first identification, characterization, and functional validation of a human miRNA promoter in stem cells. This study opens up new avenues to further investigate the upstream transcriptional regulation of the miR302-367 cluster and to dissect how these miRNAs integrate in the complex molecular network conferring stem cell properties to ESCs.

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. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.

RNase/Anti-RNase Activities of the Bacterial parD Toxin-Antitoxin System

The bacterial parD toxin-antitoxin system of plasmid R1 encodes two proteins, the Kid toxin and its cognate antitoxin, Kis. Kid cleaves RNA and inhibits protein synthesis and cell growth in Escherichia coli. Here, we show that Kid promotes RNA degradation and inhibition of protein synthesis in rabbit reticulocyte lysates. These new activities of the Kid toxin were counteracted by the Kis antitoxin and were not displayed by the KidR85W variant, which is nontoxic in E. coli. Moreover, while Kid cleaved single- and double-stranded RNA with a preference for UAA or UAC triplets, KidR85W maintained this sequence preference but hardly cleaved double-stranded RNA. Kid was formerly shown to inhibit DNA replication of the ColE1 plasmid. Here we provide in vitro evidence that Kid cleaves the ColE1 RNA II primer, which is required for the initiation of ColE1 replication. In contrast, KidR85W did not affect the stability of RNA II, nor did it inhibit the in vitro replication of ColE1. Thus, the endoribonuclease and the cytotoxic and DNA replication-inhibitory activities of Kid seem tightly correlated. We propose that the spectrum of action of this toxin extends beyond the sole inhibition of protein synthesis to control a broad range of RNA-regulated cellular processes.

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

Abstract Hepatitis C virus (HCV) infection is one of the worlds 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. The selection protocol was applied to a population of more than 1015 variants of an anti-hepatitis C virus ribozyme covalently linked to an aptamer motif. The ribozyme was directed against positions 357 to 369 of the HCV IRES, and the cleavage substrate was a 691-nucleotide-long RNA fragment that comprises the entire HCV IRES domain. After six selection cycles, seven groups of RNA variants were identified. A representative of each group was tested for its capacity to inhibit IRES activity using in vitro translation assays. All selected RNAs promoted significant inhibition, some by as much as 95%.

The functional RNA domain 5BSL3.2 within the NS5B coding sequence influences hepatitis C virus IRES-mediated translation.

Hepatitis C virus (HCV) translation is mediated by an internal ribosome entry site (IRES) located at the 5′ end of the genomic RNA. The 3′ untranslatable region (3′UTR) stimulates translation by the recruitment of protein factors that simultaneously bind to the 5′ end of the viral genome. This leads to the formation of a macromolecular complex with a closed loop conformation, similar to that described for the cap-translated mRNAs. We previously demonstrated the existence of a long-range RNA–RNA interaction involving subdomain IIId of the IRES region and the stem–loop 5BSL3.2 of the CRE element at the 3′ end of the viral genome. The present study provides evidence that the enhancement of HCV IRES-dependent translation mediated by the 3′UTR is negatively controlled by the CRE region in the human hepatoma cell lines Huh-7 and Hep-G2 in a time-dependent manner. Domain 5BSL3.2 is the major partner in this process. Mutations in this motif lead to an increase in IRES activity by up to eightfold. These data support the existence of a functional high order structure in the HCV genome that involves two evolutionarily conserved RNA elements, domain IIId in the IRES and stem–loop 5BSL3.2 in the CRE region. This interaction could have a role in the circularisation of the viral genome.

End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3′X-tail region

The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA–RNA contacts important in the establishment of viral infection. Microarray antisense oligonucelotide assays, improved dimethyl sulfate probing methods and 2′ acylation chemistry (selective 2’-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3′ end to be regulated by the internal ribosome entry site (IRES) element via direct RNA–RNA interactions. The essential cis-acting replicating element (CRE) and the 3′X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5′ terminus. Further, the structural transition in the 3′X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3′X-tail region would therefore appear to occur. The preservation of this RNA–RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle.

The folding of the hepatitis C virus internal ribosome entry site depends on the 3′-end of the viral genome

Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3′-end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3′-end. The essential IRES subdomains IIIb and IIId, and domain IV, adopted a different conformation in the presence of the cis-acting replication element and/or the 3′-untranslatable region compared to that taken up in their absence. Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element. These observations were additionally confirmed in a replication-competent RNA molecule. Significantly, protein factors are not required for these conformational differences to be made manifest. Our results suggest that a complex, direct and long-distance RNA–RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.

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. The aim of the present study was to evaluate the effect of these inhibitory RNAs on HIV-1 replication. Methods:A series of expression vectors has been constructed for the intracellular synthesis of inhibitory RNAs, differing in the promoter that drives their synthesis. These inhibitory RNAs were designed to act at two possible cleavage sites in the long terminal repeat (LTR) region and the TAR domain was chosen as a target for the antisense domain. We have evaluated the effects of different inhibitory RNAs in HIV replication via changes in p24 antigen levels. Mobility shift assays have been used to check the binding capacity of inhibitory RNAs. Results:Catalytic antisense RNA designed to target the LTR region of HIV-1 inhibited viral replication in an eukaryotic cell environment by more than 90%. The conventional hairpin and hammerhead ribozymes, however, failed to inhibit viral replication. Conclusions:The data provide preliminary evidence of a new class of inhibitory RNAs that can be used to block HIV replication. The results clearly show the importance of the ex vivo antisense effect in the inhibition achieved. A good correlation was found between the in vitro binding efficiency of the inhibitor RNA to the HIV-1 LTR and the inhibition of viral replication.

Specificity of the Hairpin Ribozyme SEQUENCE REQUIREMENTS SURROUNDING THE CLEAVAGE SITE

Substrate sequence requirements of the hairpin ribozyme have been partially defined by both mutational and in vitro selection experiments. It was considered that the best targets were those that included the N↓GUC sequence surrounding the cleavage site. In contrast to previous studies that failed to evaluate all possible combinations of these nucleotides, we have performed an exhaustive analysis of the cleavage of 64 substrate variants. They represent all possible sequence combinations of the J2/1 nucleotides except the well established G+1. No cleavage was observed with 24 sequences. C+2 variants showed little or no cleavage, whereas U+2 substrates were all cleavable. The maximal cleavage rate was obtained with the AGUC substrate. Cleavage rates of sequences HGUC (H = A, C, or U), GGUN, GGGR (R = A or G), AGUU, and UGUA were up to 5 times lower than the AGUC one. This shows that other sequences besides NGUC could also be considered as good targets. A second group of sequences WGGG (W = A or U), UGUK (K = G or U), MGAG (M = A or C), AGUA, and UGGA were cleaved between 6 and 10 times less efficiently. Furthermore, the UGCU sequence of a noncleavable viral target was mutated to AGUC resulting in a proficiently cleavable substrate by its cognate hairpin ribozyme. This indicates that our conclusions may be extrapolated to other hairpin ribozymes with different specificity.