Showing papers on "Small hairpin RNA published in 2000"

Total silencing by intron-spliced hairpin RNAs

Abstract: Post-transcriptional gene silencing (PTGS), a sequence-specific RNA degradation mechanism inherent in many life-forms, can be induced in plants by transforming them with either antisense1 or co-suppression2 constructs, but typically this results in only a small proportion of silenced individuals. Here we show that gene constructs encoding intron-spliced RNA with a hairpin structure can induce PTGS with almost 100% efficiency when directed against viruses or endogenous genes. These constructs could prove valuable in reverse genetics, genomics, engineering of metabolic pathways and protection against pathogens.

Heritable gene silencing in Drosophila using double-stranded RNA.

Abstract: RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants, nematodes, Drosophila, zebrafish, and mice. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene. Rapid degradation of mRNA in affected cells blocks gene expression. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.

The tetranucleotide UCAY directs the specific recognition of RNA by the Nova K-homology 3 domain

Abstract: The Nova family of proteins are target antigens in the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia and contain K-homology (KH)-type RNA binding domains. The Nova-1 protein has recently been shown to regulate alternative splicing of the α2 glycine receptor subunit pre-mRNA by binding to an intronic element containing repeats of the tetranucleotide UCAU. Here, we have used selection-amplification to demonstrate that the KH3 domain of Nova recognizes a single UCAY element in the context of a 20-base hairpin RNA; the UCAY tetranucleotide is optimally presented as a loop element of the hairpin scaffold and requires protein residues C-terminal to the previously defined KH domain. These results suggest that KH domains in general recognize tetranucleotide motifs and that biological RNA targets of KH domains may use either RNA secondary structure or repeated sequence elements to achieve high affinity and specificity of protein binding.

Replicon-based vectors of positive strand RNA viruses

Abstract: Vectors based on self-replicating RNAs (replicons) of positive strand RNA viruses are becoming powerful tools for gene expression in mammalian cells and for the development of novel antiviral and anticancer vaccines. A relatively small genome size and simple procedure allow rapid generation of recombinants. Cytoplasmic RNA amplification eliminates nuclear involvement and leads to extremely high levels of gene expression, and continuous synthesis of double stranded RNA results in induction of enhanced immune responses, making these vectors unique among other gene expression systems. Both cytopathic replicon vectors allowing short-term transient expression, and non-cytopathic replicon vectors allowing long-term stable expression, are now available with the choice of vector depending on particular applications.

Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences

Abstract: Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (−sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with −sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic −sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3′ end of the −sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.

RNA double cleavage by a hairpin-derived twin ribozyme

Abstract: The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.

A structured RNA motif is involved in correct placement of the tRNA(3)(Lys) primer onto the human immunodeficiency virus genome.

Abstract: The replication cycle of human immunodeficiency virus type 1 (HIV-1) and other retroviruses is characterized by reverse transcription of the viral RNA genome into a double-stranded DNA, which subsequently becomes integrated into the host cell genome (42). This process is mediated by the virion-associated enzyme reverse transcriptase (RT), and the cellular tRNA3Lys molecule is used as a primer by HIV-1 (35). The tRNA primer binds with its 3′-terminal 18 nucleotides (nt) to a complementary sequence in the viral genome, the primer-binding site (PBS), which is located in the untranslated leader region of the viral genome (Fig. ​(Fig.1A).1A). Besides the complementarity between the PBS and the 3′ end of tRNA3Lys, annealing of the primer has been proposed to be stimulated by additional base-pairing interactions between other parts of the tRNA molecule and viral sequences flanking the PBS (31). FIG. 1 Annealing of the tRNA3Lys primer to the PBS of the HIV-1 RNA genome. (A) The tRNA3Lys primer binds with its 3′ terminus to the complementary sequence of the PBS to form an 18-bp duplex that is shown in detail (PBS sequence is marked in grey). ... Extensive secondary structure in the 5′ untranslated leader region of the HIV-1 genome has been suggested by electron microscopy, replication studies with mutant viruses, and biochemical RNase probing studies (3, 11, 17, 21, 22, 37). These results, combined with phylogenetic analyses and computer-assisted structure prediction, led to a model of the secondary RNA structure of the complete leader region of the HIV-1 genome (4). According to this model, the PBS is flanked by an upstream small stem-loop structure, the U5-PBS hairpin (Fig. ​(Fig.1A).1A). This HIV-1 hairpin structure was modeled primarily based on the fact that phylogenetic analysis of different HIV and simian immunodeficiency viruses (SIV) demonstrated a conservation of the hairpin structure, despite considerable divergence in sequence (5, 7). A striking feature of the U5-PBS hairpin of different HIV and SIV isolates is that part of the PBS sequence is involved in base pairing (Fig. ​(Fig.1B).1B). Several RNA secondary structures in the leader RNA have been reported to regulate important viral replication steps of HIV-1; examples are transcriptional transactivation by Tat (8, 20, 30), mRNA polyadenylation (16, 28), and dimerization of the viral RNA genome (9, 12, 38). A stem-loop structure at a similar position as the U5-PBS hairpin of HIV-1 was predicted for Rous sarcoma virus. This structure is required for efficient initiation of reverse transcription in Rous sarcoma virus (2, 13). In addition, an interaction between U5 RNA and sequences of the primer tRNA has been proposed (1) and was confirmed recently by RNA structure probing studies (37a). A detailed structure has also been proposed for the HIV-1 RNA-tRNA3Lys complex based on biochemical experiments (24, 25a). Several sequences in the U5 region upstream of the PBS were suggested to interact with different parts of the tRNA3Lys primer. According to this model, base pairing occurs between the U-rich anticodon loop of tRNA3Lys and the A-rich loop of the U5-PBS hairpin. These combined observations suggest a specific role for the U5-PBS hairpin structure in the process of reverse transcription. To study the role of the U5-PBS hairpin in the viral replication cycle, we introduced mutations in this structured RNA motif of the HIV-1 genome. Stabilization or destabilization of the U5-PBS hairpin significantly reduced virus replication. Analysis of revertant viruses, obtained through prolonged culturing of the mutant viruses, revealed that the thermodynamic stability of the hairpin has to stay within narrow limits for efficient HIV-1 replication. Biochemical assays demonstrated the involvement of the U5-PBS hairpin in the correct placement of the tRNA3Lys primer onto the viral genome.

Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA

Abstract: The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5′-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3′ side, which contains the Shine–Dalgarno sequence. Here, we show by in vitro gp43–RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine–Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator’s hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5′-AC-3′ in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.

RNA transformation vectors derived from an uncapped single-component RNA virus

Abstract: This invention is directed to a plus strand RNA viral vector for transformation of a host organism with a foreign RNA, and expression of said foreign RNA. The foreign RNA is inserted into an infective RNA viral segment containing cis-acting viral replication elements, and allowed to infect the host organism. The RNA vector is modified to obtain infectivity by not incorporating a cap at the 5′ end of the genome. The modified RNA is able to tolerate the exogenous RNA segment without disrupting the replication of the modified RNA, in the absence of a trans-acting viral replication element in a single component plant virus host cell.