262,335 research outputs found
U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus
No full textThe in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector
Use of adenoviral VAI small RNA as a carrier for cytoplasmic delivery of ribozymes
No full textThe in vivo effectiveness of therapeutic RNAs, like antisense molecules and ribozymes, relies on several features: RNA molecules need to be expressed at high levels in the correct cellular compartment as stable and active molecules. The exploitation of "natural" small RNA coding genes as expressing cassettes gives high chances to fulfill these requirements. We have investigated the utilization of the adenoviral VAI RNA as a cytoplasmatic carrier for expressing ribozymes against HIV-1. The conserved 5' leader sequence of HIV was chosen as a target, because it is present in all the viral transcripts and is highly conserved. Hammerhead ribozymes were substituted to different portions of the VAI RNA and the resulting chimera were tested in the in vivo system of Xenopus laevis oocytes for their level of accumulation, cellular compartmentalization, and assembly in specific ribonucleoparticles containing the La antigen. Interesting differences in the activity of the different chimera were found in both in vitro cleavage assays and S100 extracts of injected oocytes where the catalytic activity of the ribozymes in the RNP context can be analyzed
Molecular studies of the salivary glands of the African malaria vector Anopheles gambiae: a tissue-specific promoter analysis
No full textThe ribosomal protein L2 in S. cerevisiae controls the level of accumulation of its own mRNA
No full textThe expression of the yeast L2 r-protein gene is controlled at the level of mRNA accumulation. The product of the gene appears to participate in this regulation by an autogenous feedback mechanism. This control does not operate at the level of transcription but instead affects L2 mRNA accumulation. This autogenous regulation of mRNA accumulation provides an interesting analogy to the autogenous translational regulation of r-proteins in Escherichia coli
Fibrillarin binds directly and specifically to U16 box C/D snoRNA
No full textEukaryotic nucleoli contain a large family of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs) that are involved in processing and site-specific methylation of pre-rRNA, Several proteins have been reported to be common factors of box C/D snoRNPs in lower and higher eukaryotes; nevertheless none of them has been clearly shown to directly interact with RNA. We previously identified in Xenopus laevis, by means of UV crosslinking in vivo, two proteins associated with box C/D snoRNAs, fibrillarin and p68, Here we show that fibrillarin interacts directly and specifically with the U16 box CID snoRNA in a X. laevis oocyte nuclear extract and that it does not require p68 for binding. Specific binding is also obtained with a recombinant fibrillarin demonstrating that the protein is able to bind directly and specifically to U16 snoRNA by itself
A novel small nucleolar RNA (U16) is encoded inside a ribosomal protein intron and originates by processing of the pre-mRNA
No full textWe report that the third intron of the L1 ribosomal protein gene of Xenopus laevis encodes a previously uncharacterized small nucleolar RNA that we called U16. This snRNA is not independently transcribed; instead it originates by processing of the pre-mRNA in which it is contained. Its sequence, localization and biosynthesis are phylogenetically conserved: in the corresponding intron of the human L1 ribosomal protein gene a highly homologous region is found which can be released from the pre-mRNA by a mechanism similar to that described for the amphibian U16 RNA. The presence of a snoRNA inside an intron of the L1 ribosomal protein gene and the phylogenetic conservation of this gene arrangement suggest an important regulatory/functional link between these two components
The Rev protein is able to transport to the cytoplasm small nucleolar RNAs containing a Rev binding element
No full textSmall nucleolar RNAs (snoRNAs) were utilized to express Rev-binding sequences inside the nucleolus and to test whether they are substrates for Rev binding and transport. We show that U16 snoRNA containing the minimal binding site for Rev stably accumulates inside the nucleolus maintaining the interaction with the basic C/D snoRNA-specific factors. Upon Rev expression, the chimeric RNA is exported to the cytoplasm, where it remains bound to Rev in a particle devoid of snoRNP-specific factors. These data indicate that Rev can elicit the functions of RNA binding and transport inside the nucleolus
Two different snoRNAs are encoded in introns of amphibian and human L1 ribosomal protein genes
No full textWe previously reported that the third intron of the X.laevis L1 ribosomal protein gene encodes for a snoRNA called U16. Here we show that four different introns of the same gene contain another previously uncharacterized snoRNA (U18) which is associated with fibrillarin in the nucleolus and which originates by processing of the pre-mRNA. The pathway of U18 RNA release from the pre-mRNA is the same as the one described for U16: primary endonucleolytic cleavages upstream and downstream of the U18 coding region produce a pre-U18 RNA which is subsequently trimmed to the mature form. Both the gene organization and processing of U18 are conserved in the corresponding genes of X.tropicalis and H.sapiens. The L1 gene thus has a composite structure, highly conserved in evolution, in which sequences coding for a ribosomal protein are intermingled with sequences coding for two different snoRNAs. The nucleolar localization of these different components suggests some common function on ribosome biosynthesis
Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIV ribozymes in a human T lymphoblastoid cell line
No full textHuman immunodeficiency virus (HIV) infection represents one of the most challenging systems for gene therapy. Thanks to the extended knowledge of the molecular biology of the HIV life cycle, many different strategies have been developed including transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes, and intracellular antibody fragments. In this paper, we have tested in a human T lymphoblastoid cell line the antiviral activity of ribozymes specifically designed to co-localize inside the nucleus with the Rev pre-mRNA before it is spliced and transported to the cytoplasm. This result was obtained by inserting the ribozyme in the spliceosomal U1 small nuclear RNA (snRNA) and in a derivative that has perfect complementarity with the 5' splice site of the Rev pre-mRNA. These ribozymes were tested in human T cell clones and were shown to be very efficient in inhibiting viral replication. Not only were the p24 levels in the culture medium drastically reduced but so were the intracellular HIV transcripts. Control disabled ribozymes enabled us to show the specificity of the ribozyme activity. Therefore, these constructs have potential utility for gene therapy of HIV-1 infection
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