1,721,043 research outputs found
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
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
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
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
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
Ribosomal protein production in normal and anucleolate Xenopus embryos: regulation at the posttranscriptional and translational levels
No full textWe have studied the regulation of ribosomal protein (r-protein) synthesis in Xenopus anucleolate mutants, which lack the genes for rRNA. The accumulation of mRNA for the two r-proteins analyzed parallels the controls up to stage 30. This mRNA is mobilized onto polysomes and is translated as in normal embryos, but r-proteins are unstable in the absence of rRNA to assemble with. A translational control of rp-mRNA distribution between polysomes and mRNPs is observed, but this is not due to an autogenous regulation by r-proteins. After stage 30 the amount of rp-mRNA declines specifically in the mutants because the transcripts are unstable. Considering the temporal correlation between this event and the onset of r-protein synthesis we suggest that an autogenous control operates at the level of transcript stability
In vitro study of processing of the intron-encoded U16 small nucleolar RNA in Xenopus laevis
No full textIt was recently shown that a new class of small nuclear RNAs is encoded in introns of protein-coding genes and that they originate by processing of the pre-mRNA in which they are contained. Little is known about the mechanism and the factors involved in this new type of processing. The L1 ribosomal protein gene of Xenopus laevis is a well-suited system for studying this phenomenon: several different introns encode for two small nucleolar RNAs (snoRNAs; U16 and U18). In this paper, we analyzed the in vitro processing of these snoRNAs and showed that both are released from the pre-mRNA by a common mechanism: endonucleolytic cleavages convert the pre-mRNA into a precursor snoRNA with 5' and 3' trailer sequences. Subsequently, trimming converts the pre-snoRNAs into mature molecules. Oocyte and HeLa nuclear extracts are able to process X. laevis and human substrates in a similar manner, indicating that the processing of this class of snoRNAs relies on a common and evolutionarily conserved mechanism. In addition, we found that the cleavage activity is strongly enhanced in the presence of Mn2+ ions
The role of long noncoding RNAs in the epigenetic control of gene expression
No full textRecent advances in the methodologies employed to deeply analyse the complexity of transcriptomes have unveiled the existence of a new class of transcripts, long noncoding RNAs (lncRNAs). A significant amount of effort has been dedicated to the study of lncRNAs, and a large body of evidence now exists indicating their relevant role in different regulatory steps of gene expression. Given the role of epigenetics in disease development and progression, this Minireview focuses on lncRNAs involved in epigenetic control and provides an overview of the mechanisms used to guide epigenetic-modifying complexes to adjacent (cis-acting) or independent (trans-acting) genomic loci. Furthermore, it describes the activities of these transcripts in controlling the formation and spreading of heterochromatin domains. Just as other RNA molecules have found therapeutic application, though much remains to be elucidated about the structure and function of these lncRNAs, they too could hold potential as biomarkers, targets, and therapeutic agents
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