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At least 631 records · Page 35Linked to original sources
PubMed2005

RNA silencing in Drosophila.

Knowledge of the role of RNA in affecting gene expression has expanded in the past several years. Small RNAs serve as homology guides to target messenger RNAs for destruction at the post-transcriptional level in the experimental technique known as RNA interference and in the silencing of some transgenes. These small RNAs are also involved in sequence-specific targeting of chromatin modifications for transcriptional silencing of transgenes, transposable elements, heterochromatin and some cases of Polycomb-mediated gene silencing. RNA silencing processes in Drosophila are described.

Animals↗
PubMed2003

Dyskeratosis congenita: its link to telomerase and aplastic anaemia.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome exhibiting considerable clinical and genetic heterogeneity. X-linked recessive, autosomal dominant and autosomal recessive forms are recognised. The gene mutated in X-linked DC (DKC1) encodes a highly conserved nucleolar protein called dyskerin. Dyskerin associates with the H/ACA class of small nucleolar RNAs which are important in guiding the conversion of uracil to pseudouracil in ribosomal RNA. Dyskerin also associates with the RNA component of telomerase (hTR) which is important in the maintenance of telomeres. Mutations in hTR were recently demonstrated in patients with autosomal dominant DC and in a subset of patients with aplastic anaemia (AA) but without other diagnostic features of DC. This discovery demonstrates that both DC and a subset of AA are due to a defect in telomerase. The link between DC and AA and in turn to defective telomerase suggests that treatments directed at correction of telomerase activity might benefit DC/AA patients who do not respond to conventional therapy.

Anemia, Aplastic↗
PubMed2005

snRNA and heterochromatin formation are involved in DNA excision during macronuclear development in stichotrichous ciliates.

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant "X" is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

Animals↗
PubMed2004

DNA methylation and epigenetics.

In many eukaryotes, including plants, DNA methylation provides a heritable mark that guides formation of transcriptionally silent heterochromatin. In plants, aberrant RNA signals direct DNA methylation to target sequences, sometimes appropriately and sometimes inappropriately. This chapter discusses the generation of RNA signals for epigenetic changes, the factors that mediate those changes, and some of the consequences of those changes for plant gene expression and genome integrity.

DNA Methylation↗
PubMed2005

Construction of an effective m1 GS ribozyme targeting HCMV UL97 mRNA segment in vitro.

A sequence-specific M1GS ribozyme (M1-T3) was constructed by covalently linking an oligonucleotide (guide sequence,GS) to the 3' terminus of M1 RNA ,the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the mRNA sequence encoding a protein kinase (UL97) of HCMV and could effectively cleave the mRNA segment in vitro. Further studies about the significance of some structural elements in the M1 GS (e.g. the 3' CCA tail sequence and a bridge sequence between the 3' terminus of M1 RNA and the 5' terminus of the GS) were carried out. The results showed that the bridge sequence of 88 nucleotides in a mutated M1 GS (i.e. M1-T3*) dramatically increased the cleavage activity to the substrate in vitro. Moreover, the 3'CCA tail sequence was confirmed to be a necessary element for the cleavage activity of M1 GS ribozyme. These data we got in the study will help in understanding the interaction between the M1 GS RNA and its substrate,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.

5' Untranslated Regions↗
PubMed2002

RNA and RNA-protein complexes as targets for therapeutic intervention.

Today, the majority of pharmaceuticals developed to treat cancers and viral/bacterial infections target cellular, bacterial or viral proteins known to be associated with a given pathology. Although proteins are the focus of most current drug discovery efforts, exciting new research has recently begun which aims to exploit ribonucleic acid (RNA) and RNP particles as novel targets for pharmaceutical development. These RNA-targeted research efforts have been fueled by an increased appreciation for the central role played by RNA and RNA-protein interactions in many biological processes and diseases, together with a better understanding of RNA structure and an improvement in biophysical/biochemical techniques available to study RNA. As for protein targets, genome sequencing is greatly accelerating the identification of human and microbial RNA transcripts for targeted drug discovery. With this explosion in the number of potential RNA and RNP targets, the effective development of specific small molecule RNA-based drugs requires robust and general approaches for detecting and quantifying RNA-ligand interactions, which can be used as high-throughput screens (HTS) and for obtaining rapid structural information to guide rational drug design. In this review, an overview of the potential for therapeutic intervention based on RNA and RNP targets is presented, together with recent efforts to develop generally useful nuclear magnetic resonance (NMR) and fluorescence binding assays for screening and optimizing drugs aimed at RNA and RNP targets.

Animals↗
PubMedDate not supplied

The impact of molecular biology in the diagnosis and management of viral hepatitis.

In recent years molecular biology techniques allowed an extraordinary progress in knowledge of hepatitis viruses. New informations have accumulated on the mechanisms of virus replication and on the pathogenesis of liver damage. In the clinical setting, simple noninvasive methods that use the same technological approach provide a useful diagnostic tool for infections of hepatitis B virus (HBV) and hepatitis delta virus (HDV). Using these tests we may define whether a carrier of hepatitis B surface antigen has a liver damage due to HBV or HDV or to other, non-virological causes. Detection of HBV-DNA and HDV-RNA in serum and liver can also be used as a guide for prognosis and therapy.

Animals↗
PubMed2006

RNA reprogramming of alpha-mannosidase mRNA sequences in vitro by myxomycete group IC1 and IE ribozymes.

Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated alpha-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant alpha-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated alpha-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4-P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing.

Animals↗
PubMed2026

Genome-guided stage- and tissue-resolved transcriptome analysis of Serrodes campana identifies sex-biased antennal expression and candidate chemosensory-related genes.

Serrodes campana is an erebid moth of ecological and forestry relevance; its larvae are mainly associated with the soapberry tree, Sapindus mukorossi, whereas adults exhibit fruit-piercing behavior. However, stage- and tissue-resolved transcriptomic resources for this species remain limited. Here, using a chromosome-level reference genome, we performed a genome-guided transcriptome analysis of S. campana based on 12 RNA-seq libraries representing major developmental stages and key adult tissues. Global transcriptomic analyses revealed pronounced transcriptional differentiation across developmental stages and tissue types. Tissue-enriched gene sets and functional enrichment analyses identified distinct molecular signatures associated with developmental, sensory, and pheromone-associated tissues. Comparative analysis of female and male antennae further revealed sex-biased expression of several candidate chemosensory-related genes. Among 153 curated chemosensory-related candidate genes, most odorant receptor genes showed strong antennal enrichment, whereas other major chemosensory gene families displayed broader but still tissue-preferential expression patterns. In addition, an exploratory comparison of female terminal abdominal gland tissue and male terminal abdominal coremata revealed divergent expression profiles and highlighted candidate genes potentially associated with pheromone-related physiology, reproduction, and tissue-specific signaling. Together, this study provides the first genome-guided stage- and tissue-resolved transcriptomic resource for S. campana and offers a useful foundation for future studies of chemosensory detection, sex-biased gene expression, and pheromone-associated biology in this species.

Animals↗
PubMed1987

New RNA-mediated reactions by yeast mitochondrial group I introns.

The group I self-splicing reaction is initiated by attack of a guanosine nucleotide at the 5' splice site of intron-containing precursor RNA. When precursor RNA containing a yeast mitochondrial group I intron is incubated in vitro under conditions of self-splicing, guanosine nucleotide attack can also occur at other positions: (i) the 3' splice site, resulting in formation of a 3' exon carrying an extra added guanosine nucleotide at its 5' end; (ii) the first phosphodiester bond in precursor RNA synthesized from the SP6 bacteriophage promoter, leading to substitution of the first 5'-guanosine by a guanosine nucleotide from the reaction mixture; (iii) the first phosphodiester bond in already excised intron RNA, resulting in exchange of the 5' terminal guanosine nucleotide for a guanosine nucleotide from the reaction mixture. An identical sequence motif (5'-GAA-3') occurs at the 3' splice site, the 5' end of SP6 precursor RNA and at the 5' end of excised intron RNA. We propose that the aberrant reactions can be explained by base-pairing of the GAA sequence to the Internal Guide Sequence. We suggest that these reactions are mediated by the same catalytic centre of the intron RNA that governs the normal splicing reactions.

Base Sequence↗
PubMed1993

Antisense oligoribonucleotides and RNase P. A great potential.

This paper presents the possible--but at present mostly hypothetical--applications of RNase P for the specific inactivation of target RNAs. The natural substrates for RNase P are pre-tRNAs. The enzyme can also recognize and cleave smaller model substrates. These are simple hairpins for bacterial RNase P whereas the requirements for eukaryotic RNase P are more complex and less well understood. It is possible to split the RNase P substrates into two separate RNA molecules. One part of the split substrate RNA contains the RNase P cleavage site and the 5'-terminal half of the acceptor stem, embedded in a large target RNA. The other part of the substrate RNA provides the 3'-terminal half of the acceptor stem and it serves as antisense sequence ('external guide sequence'). Both parts can hybridize and reconstitute a functional substrate for RNase P; this results in cleavage of the target RNA by RNase P. The properties of this system are presented and advantages and problems discussed.

Animals↗
PubMed2002

Characterization of the interaction of Bacillus subtilis PyrR with pyr mRNA by site-directed mutagenesis of the protein.

The Bacillus subtilis PyrR protein regulates transcriptional attenuation of the pyrimidine nucleotide (pyr) operon by binding in a uridine nucleotide-dependent manner to specific sites on pyr mRNA and stabilizing a secondary structure of the downstream RNA that favors termination of transcription. The high-resolution structure of unliganded PyrR was used to guide site-directed mutagenesis of 12 amino acid residues that were thought likely to be involved in the binding of RNA. Missense mutations were constructed and evaluated for their effects on regulation of pyr genes in vivo and their uracil phosphoribosyltransferase activity, which is catalyzed by wild-type PyrR. A substantial fraction of the mutant PyrR proteins did not have native structures, but eight PyrR mutants were purified and characterized physically, for their uracil phosphoribosyltransferase activity and for their ability to bind pyr RNA in vitro. On the basis of these studies Thr-18, His-22, Arg-141, and Arg-146 were implicated in RNA binding. Arg-27 and Lys-152 were also likely to be involved in RNA binding, but Gln substitution mutations in these residues may have altered their subunit-subunit interactions slightly. Arg-19 was implicated in pyr regulation, but a specific role in RNA binding could not be demonstrated because the R19Q mutant protein could not be purified in native form. The results confirm a role in RNA binding of a positively charged face of PyrR previously identified from the crystallographic structure. The RNA binding residues lie in two sequence segments that are conserved in PyrR proteins from many species.

Amino Acid Sequence↗
PubMed2005

The silent revolution: RNA interference as basic biology, research tool, and therapeutic.

RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression. In primitive organisms, RNAi protects the genome from viruses and other insertable genetic elements and regulates gene expression during development. The antisense (guide) strand of short double-stranded RNAs is incorporated into an RNA-induced silencing complex that can either suppress protein expression or direct degradation of messenger RNAs that contain homologous sequence(s). The discovery that RNAi works in mammalian cells has sparked intense investigation into its role in normal mammalian cell function, its use as a tool to understand or screen for genes functioning in cellular pathways in healthy and diseased cells and animals, and its potential for therapeutic gene silencing. RNAi may provide an important new therapeutic modality for treating infection, cancer, neurodegenerative disease, and other illnesses, although in vivo delivery of small interfering RNAs into cells remains a significant obstacle.

Animals↗
PubMed2000

Characterisation of the U83 and U84 small nucleolar RNAs: two novel 2'-O-ribose methylation guide RNAs that lack complementarities to ribosomal RNAs.

In eukaryotic cells, the site-specific 2'- O -ribose methylation of ribosomal RNAs (rRNAs) and the U6 spliceosomal small nuclear RNA (snRNA) is directed by small nucleolar RNAs (snoRNAs). The C and D box-containing 2'- O -methylation guide snoRNAs select the correct substrate nucleotide through formation of a long 10-21 bp interaction with the target rRNA and U6 snRNA sequences. Here, we report on the characterisation of two novel mammalian C/D box snoRNAs, called U83 and U84, that contain all the elements that are essential for accumulation and function of 2'- O -methylation guide snoRNAs. However, in contrast to all of the known 2'- O -methylation guide RNAs, the human, mouse and pig U83 and U84 snoRNAs feature no antisense elements complementary to rRNA or U6 snRNA sequences. The human U83 and U84 snoRNAs are not associated with maturing nucleolar pre-ribosomal particles, suggesting that they do not function in rRNA biogenesis. Since artificial substrate RNAs complementary to the evolutionarily conserved putative substrate recognition motifs of the U83 and U84 snoRNAs were correctly 2'- O -methylated in the nucleolus of mouse cells, we suggest that the new snoRNAs act as 2'- O -methylation guides for cellular RNAs other then rRNAs and the U6 snRNA.

Animals↗
PubMed2001

RNA editing-associated protein 1 is an RNA binding protein with specificity for preedited mRNA.

RNA editing in the mitochondria of kinetoplastids involves the addition and deletion of uridines at specific sites as directed by guide RNAs (gRNAs). Ample evidence shows that ribonucleoprotein (RNP) complexes carry out this posttranscriptional processing. One component of RNA editing complexes is REAP-1, a protein of previously unknown function found primarily in mRNA containing editing complexes. We now show that REAP-1 is an RNA binding protein and map the binding activity to the amino-terminal third of the protein. REAP-1 binds to poly(G) and single-stranded guanosine rich RNAs. Data presented here demonstrates that preedited RNAs are the preferred substrate for REAP-1. The results suggest a model in which the role of REAP-1 is to bring preedited mRNAs into the editing complex.

Animals↗
PubMed2001

RNA interference is mediated by 21- and 22-nucleotide RNAs.

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.

Animals↗
PubMed2006

A large collection of compact box C/D snoRNAs and their isoforms in Euglena gracilis: structural, functional and evolutionary insights.

In the domains Eucarya and Archaea, box C/D RNAs guide methylation at the 2'-position of selected ribose residues in ribosomal RNA (rRNA). Those eukaryotic box C/D RNAs that have been identified to date are larger and more variable in size than their archaeal counterparts. Here, we report the first extensive identification and characterization of box C/D small nucleolar (sno) RNAs from the protist Euglena gracilis. Among several unexpected findings, this organism contains a large assortment of methylation-guide RNAs that are smaller and more uniformly sized than those of other eukaryotes, and that consist of surprisingly few double-guide RNAs targeting sites of rRNA modification. Our comprehensive examination of the modification status of E.gracilis rRNA indicates that many of these box C/D snoRNAs target clustered methylation sites requiring extensive, overlapping guide RNA/rRNA pairings. An examination of the structure of the RNAs, in particular the location of the functional guide elements, suggests that the distances between adjacent box elements are an important factor in determining which of the potential guide elements is used to target a site of O(2')-methylation.

Animals↗
PubMed2003

R2D2 leads the silencing trigger to mRNA's death star.

During RNA interference (RNAi), Dicer generates short interfering RNAs (siRNAs), which then guide target mRNA cleavage by the RISC complex. Now, Liu et al. identify R2D2, a Dicer-associated protein that is important for siRNA incorporation into RISC, thus linking the initiation and execution phases of RNAi.

Animals↗