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ABSTRACT: 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.
Nucleic Acids Research 10/2012; · 8.03 Impact Factor
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ABSTRACT: de Ajalvir km 4, 28850 Madrid, Spain and 4 Centro de Investigació n Biomé dica en Red de enfermedades hepá ticas y digestivas (CIBERehd), Spain ABSTRACT Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3 0 -end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibil-ity using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3 0 -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 0 -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 re-activity 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.
Nucleic Acids Research 10/2012; 40(22):11697. · 8.03 Impact Factor
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ABSTRACT: 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 mechanisms by which this process occurs have been extensively studied and begin to be much better understood. They involve a network of complex pathways that use intrinsic features of the target mRNA, like stability, to control its relative abundance in the cytoplasm. Thus, the analysis of the mRNA stability and abundance is essential to properly undertake gene expression studies. This chapter describes the ribonuclease protection assay, a widely accepted approach to evaluate the quality and amount of a target mRNA. This technique displays a higher sensitivity than classical Northern blot analysis and may be used either individually or in combination with other quantitative methods, such as quantitative reverse-transcription PCR, as complementary procedures rendering more complete and reliable information on gene expression.
Methods in molecular biology (Clifton, N.J.) 01/2012; 809:491-503.
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ABSTRACT: 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. The promoter activity was functionally validated in hESCs, opening up new avenues to further investigate how these miRNA molecules fit in the complex molecular network conferring "stemness" properties to hESCs. The physiological roles of specific miRNA-mRNA interactions remain largely unknown. Here, we investigated putative miR-302-367 mRNA targets in hESCs, potentially relevant for ESC biology. We found that the Nodal inhibitors Lefty1 and Lefty2 are post-transcriptionally targeted by miR-302s in hESCs. Functional analyses indicate that miR-302s negatively modulate the level of lefties, and become upstream regulators of the TGFβ/Nodal pathway, functioning via Smad-2/3 signaling. Overexpression of the miR-302-367 cluster in hESCs causes a delay in early hESC differentiation, as measured by enhanced levels of ESC-specific transcription factors, coupled to a faster teratoma formation in mice transplanted with miR-302-367-expressing hESCs and a concomitant impairment of germ layer specification, displaying robust decreased levels of early mesodermal, endodermal, and ectodermal specific markers. These findings suggest that Lefty is negatively modulated by miR-302s in hESCs, which plays an important role in maintaining the balance between pluripotency and germ layer specification.
The FASEB Journal 01/2011; 25(5):1497-508. · 5.71 Impact Factor
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ABSTRACT: 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 inhibitor RNA interferes with the formation of a translationally active complex, stalling its progression at the level of 80S particle formation. This action is likely related to the effective and specific blocking of HCV IRES-dependent translation achieved in Huh-7 cells. The inhibitor HH363-50 also reduces HCV RNA levels in a subgenomic replicon system. The present findings suggest that HH363-50 could be an effective anti-HCV compound and highlight the possibilities of antiviral agents based on RNA molecules.
Journal of General Virology 04/2009; 90(Pt 7):1659-69. · 3.36 Impact Factor
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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.
Molecular and cellular biology 09/2008; 28(21):6609-19. · 6.06 Impact Factor
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ABSTRACT: 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. The main features of the target sequence are also examined and other procedures or modifications of interest are
also discussed.
02/2008: pages 327-338;
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ABSTRACT: 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). This represents a significant improvement of the inhibitory efficiency of the ribozyme, suggesting there is an additive inhibitory effect on HIV-1 replication by the catalytic and decoy domains. This strategy could be used to create new inhibitor RNAs with enhanced in vivo performance.
RNA biology 05/2005; 2(2):75-9. · 5.56 Impact Factor
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ABSTRACT: 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. The selection protocol was applied to a population of more than 10(15) 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%.
Biological Chemistry 03/2005; 386(2):183-90. · 2.96 Impact Factor
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ABSTRACT: 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. The hairpin ribozyme is attached to the target mRNA, and is adequately randomized to generate a population representing all possible substrate specificities. The selection procedure yields information on the best target sites, and provides information about optimal ribozyme sequences. Thus, this method helps in the rational design of efficient hairpin ribozymes for targeting purposes, and avoids trial-and-error assays usually associated with theoretical ribozyme design.
Methods in molecular biology (Clifton, N.J.) 02/2004; 252:313-25.
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ABSTRACT: 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. The main features of the target sequence are also examined and other procedures or modifications of interest are also discussed.
Methods in molecular biology (Clifton, N.J.) 02/2004; 252:327-38.
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ABSTRACT: 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.
FEMS Microbiology Reviews 05/2003; 27(1):75-97. · 10.96 Impact Factor
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ABSTRACT: 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. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.
Antisense and Nucleic Acid Drug Development 03/2002; 12(1):1-9.
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ABSTRACT: 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.
Journal of Biological Chemistry 10/1999; 274(41):29376-29380. · 4.77 Impact Factor
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ABSTRACT: The identification of proficient target sites within long RNA molecules, as well as the most efficient ribozymes for each, is a major concern for the use of ribozymes as gene suppressers. In vitro selection methods using combinatorial libraries are powerful tools for the rapid elucidation of interactions between macromolecules, and have been successfully used for different types of ribozyme study. This paper describes a new method for selecting effective target sites within long RNAs using a combinatorial library of self-cleaving hairpin ribozymes that includes all possible specificities. The method also allows the identification of the most appropriate ribozyme for each identified site. Searching for targets within the lacZ gene with this strategy yielded a clearly accessible site. Sequence analysis of ribozymes identified two variants as the most appropriate for this site. Both selected ribozymes showed significant inhibitory activity in the cell milieu.
