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At least 487 records · Page 27Linked to original sources
PubMed2000

Archaeal homologs of eukaryotic methylation guide small nucleolar RNAs: lessons from the Pyrococcus genomes.

Ribose methylation is a prevalent type of nucleotide modification in rRNA. Eukaryotic rRNAs display a complex pattern of ribose methylations, amounting to 55 in yeast Saccharomyces cerevisiae and about 100 in vertebrates. Ribose methylations of eukaryotic rRNAs are each guided by a cognate small RNA, belonging to the family of box C/D antisense snoRNAs, through transient formation of a specific base-pairing at the rRNA modification site. In prokaryotes, the pattern of rRNA ribose methylations has been fully characterized in a single species so far, Escherichia coli, which contains only four ribose methylated rRNA nucleotides. However, the hyperthermophile archaeon Sulfolobus solfataricus contains, like eukaryotes, a large number of (yet unmapped) rRNA ribose methylations and homologs of eukaryotic box C/D small nucleolar ribonuclear proteins have been identified in archaeal genomes. We have therefore searched archaeal genomes for potential homologs of eukaryotic methylation guide small nucleolar RNAs, by combining searches for structured motifs with homology searches. We have identified a family of 46 small RNAs, conserved in the genomes of three hyperthermophile Pyrococcus species, which we have experimentally characterized in Pyrococcus abyssi. The Pyrococcus small RNAs, the first reported homologs of methylation guide small nucleolar RNAs in organisms devoid of a nucleus, appear as a paradigm of minimalist box C/D antisense RNAs. They differ from their eukaryotic homologs by their outstanding structural homogeneity, extended consensus box motifs and the quasi-systematic presence of two (instead of one) rRNA antisense elements. Remarkably, for each small RNA the two antisense elements always match rRNA sequences close to each other in rRNA structure, suggesting an important role in rRNA folding. Only a few of the predicted P. abyssi rRNA ribose methylations have been detected so far. Further analysis of these archaeal small RNAs could provide new insights into the origin and functions of methylation guide small nucleolar RNAs and illuminate the still elusive role of rRNA ribose methylations.

Base Sequence↗
PubMed2002

Trypanosome mitochondrial 3' terminal uridylyl transferase (TUTase): the key enzyme in U-insertion/deletion RNA editing.

A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.

Animals↗
PubMed2006

Cleavage of the siRNA passenger strand during RISC assembly in human cells.

A crucial step in the RNA interference (RNAi) pathway involves the assembly of RISC, the RNA-induced silencing complex. RISC initially recognizes a double-stranded short interfering RNA (siRNA), but only one strand is finally retained in the functional ribonucleoprotein complex. The non-incorporated strand, or 'passenger' strand, is removed during the assembly process and most probably degraded thereafter. In this report, we show that the passenger strand is cleaved during the course of RISC assembly following the same rules established for the siRNA-guided cleavage of a target RNA. Chemical modifications impairing the cleavage of the passenger strand also impair the cleavage of a target RNA in vitro as well as the silencing of a reporter gene in vivo, suggesting that passenger strand removal is facilitated by its cleavage during RISC assembly. Interestingly, target RNA cleavage can be rescued if an otherwise non-cleavable passenger strand shows a nick at the scissile phosphodiester bond, which further indicates that the cleavage event per se is not essential.

Gene Silencing↗
PubMed1992

RNA binding determinant in some class I tRNA synthetases identified by alignment-guided mutagenesis.

The N-terminal nucleotide binding folds of all 10 class I tRNA synthetases (RSs) contain characteristic conserved sequence motifs that define this class of synthetases. Sequences of C-terminal domains, which in some cases are known to interact with anticodons, are divergent. In the 676-amino acid Escherichia coli methionyl-tRNA synthetase (MetRS), interactions with the methionine tRNA anticodon are sensitive to substitutions at a specific location on the surface of the C-terminal domain of this protein of known three-dimensional structure. Although four class I synthetases of heterogeneous lengths and unknown structures are believed to be historically related to MetRS, pair-wise sequence similarities in the region of this RNA binding determinant are obscure. A multiple alignment of all sequences of three of these synthetases with all MetRS sequences suggested a location for the functional analog of the anticodon-binding site in these enzymes. We chose a member of this set for alignment-guided mutagenesis, combined with a functional analysis of mutant proteins. Substitutions within two amino acids of the site fixed by the multiple sequence alignment severely affected interactions with tRNA but not with ATP or amino acid. Multiple individual replacements at this location do not disrupt enzyme stability, indicating this segment is on the surface, as in the MetRS structure. The results suggest the location of an RNA binding determinant in each of these three synthetases of unknown structure.

Diphosphates↗
PubMed2026

CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.

Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.

CRISPR↗
PubMed2006

Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of a gRNA bound to its cognate mRNA.

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the gRNA template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex with the enzymatic machinery required. While a considerable amount of work has been done identifying the protein components of the editing complex, very little is known about how a functional editosome is assembled. In addition, the importance of RNA structure in establishing a functional editing complex is poorly understood. Work in our lab suggests that different mRNA/gRNA pairs can form similar secondary structures suggesting that a common core architecture may be important for editosome recognition and function. Using solution structure probing, we have investigated the structure of the initiating gRNA, gCYb-558, in the mRNA/gRNA complex with pre-edited apocytochrome b mRNA. Our data indicate that the stem-loop formed by the guiding region of the gRNA alone is maintained in its interaction with the pre-edited message. In addition, our data suggest that a gRNA stem-loop structure is maintained through the first few editing events by the use of alternative base-pairing with the U-tail.

Animals↗
PubMed1996

The UCUAAAC promoter motif is not required for high-frequency leader recombination in bovine coronavirus defective interfering RNA.

The 65-nucleotide leader on the cloned bovine coronavirus defective interfering (DI) RNA, when marked by mutations, has been shown to rapidly convert to the wild-type leader of the helper virus following DI RNA transfection into helper virus-infected cells. A model of leader-primed transcription in which free leader supplied in trans by the helper virus interacts by way of its flanking 5'UCUAAAC3' sequence element with the 3'-proximal 3'AGAUUUG5' promoter on the DI RNA minus strand to prime RNA replication has been used to explain this phenomenon. To test this model, the UCUAAAC element which occurs only once in the BCV 5' untranslated region was either deleted or completely substituted in input DI RNA template, and evidence of leader conversion was sought. In both cases, leader conversion occurred rapidly, indicating that this element is not required on input RNA for the conversion event. Substitution mutations mapped the crossover region to a 24-nucleotide segment that begins within the UCUAAAC sequence and extends downstream. Although structure probing of the bovine coronavirus 5' untranslated region indicated that the UCUAAAC element is in the loop of a prominent stem and thus theoretically available for base pair-directed priming, no evidence of an unattached leader early in infection that might have served as a primer for transcription was found by RNase protection studies. These results together suggest that leader conversion on the DI RNA 5' terminus is not guided by the UCUAAAC element and might arise instead from a high-frequency, region-specific, homologous recombination event perhaps during minus-strand synthesis rather than by leader priming during plus-strand synthesis.

Animals↗
PubMed2005

microRNA-guided posttranscriptional gene regulation.

microRNAs (miRNAs) form an evolutionarily conserved and highly abundant class of non-coding RNAs that are 21-24 nucleotides (nt) in length. They are processed from double-stranded (ds) RNA precursors and sequence-specifically guide posttranscriptional gene silencing. The processing steps are facilitated by members of the RNAse III enzyme family, whereas gene silencing events are mediated by members of the highly conserved Argonaute (Ago) protein family. Initially discovered in Caenorhabditis elegans, in which they are essential for proper developmental timing, hundreds of miRNAs have been discovered to date in a variety of different organisms, including plants, flies and mammals. Expression profiling approaches demonstrated that miRNAs are specifically expressed not only during embryonic development, but also during cell differentiation and other cellular events such as hormonal signaling. Although miRNAs have been the object of extensive research in recent years, very little is known about their target mRNAs. Their identification along with a comprehensive description of the miRNA/target-mRNA interaction network will add a new level to our knowledge of gene regulation and will also provide new insights into the biology of so far poorly understood diseases, including various forms of cancer.

Animals↗
PubMed2002

RNAi: nature abhors a double-strand.

In organisms as diverse as nematodes, trypanosomes, plants, and fungi, double-stranded RNA triggers the destruction of homologous mRNAs, a phenomenon known as RNA interference. RNA interference begins with the transformation of the double-stranded RNA into small RNAs that then guide a protein nuclease to destroy their mRNA targets.

Animals↗
PubMed1998

Integration of the Tetrahymena group I intron into bacterial rRNA by reverse splicing in vivo.

Horizontal gene transfer is thought to contribute to the wide distribution of group I introns among organisms. Integration of an intron into foreign RNA or DNA by reverse self-splicing, followed by reverse transcription and recombination, could lead to its transposition. Reverse self-splicing of group I introns has been demonstrated in vitro, but not in vivo. Here we report RNA-dependent integration of the Tetrahymena intron into the 23S rRNA in Escherichia coli. Analysis of products by Northern blot and reverse transcription-PCR amplification revealed precise intron insertion into a site homologous to the natural splice junction. Products are sensitive to treatment with RNase but not DNase and depend on the splicing activity of the intron. Partial reaction with 11 novel sites in the 23S RNA that are complementary to the guide sequence of the intron illustrates lower specificity than intron homing. Reverse splicing of the Tetrahymena intron in bacteria demonstrates the possibility of RNA-catalyzed transposition of group I introns in foreign hosts.

Animals↗
PubMed1999

The antibiotic viomycin as a model peptide for the origin of the co-evolution of RNA and proteins.

Viomycin is an RNA-binding peptide antibiotic which inhibits prokaryotic protein synthesis and group I intron self-splicing. This antibiotic enhances the activity of the ribozyme derived from the Neurospora crassa VS RNA, and at sub-inhibitory concentrations it induces the formation of group I intron oligomers. Here, we address the question whether viomycin exerts specificity in the promotion of RNA-RNA interactions. In an in vitro selection experiment we tested the ability of viomycin to specifically select molecules out of an RNA pool. Group I intron RNA was incubated with a pool of random sequence RNA, or with a pool of RNA molecules which had previously been enriched for viomycin-binding RNAs. Viomycin was added in order to select viomycin-binding RNAs and to guide their interaction with the intron RNA resulting in recombinant molecules. Viomycin was indeed capable of specifically selecting RNA molecules which contain viomycin-binding sites promoting recombination. These results suggest that small peptides are able to play the role of selector molecules in a putative 'RNA World' launching the co-evolution of RNA and proteins into an 'RNA-protein World'.

Anti-Bacterial Agents↗
PubMed2003

RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei.

RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by approximately 98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%-80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs.

Animals↗
PubMed2002

Immobilized small deoxyribozyme to distinguish RNA secondary structures.

The RNA folding variation due to one or more mutations leads to different RNA splicing, RNA processing, and translational controls as a result of differences in the primary and higher-ordered structures that interact with other cellular molecules. Thus, distinguishing RNA folding is one of the guides to detect the gene functions related to disease and drug responses. We found, previously, a small Ca(2+)-dependent deoxyribozyme with its site-specific RNA cleavage [Sugimoto, N., Okumoto, Y., and Ohmichi, T. (1999) J. Chem. Soc., Perkin Trans. 2, 1382-1388]. In this study, we report the potential of this deoxyribozyme as a useful tool to distinguish RNA foldings. It is found that the immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The systematic design for the target RNA hairpin loops shows that the immobilized deoxyribozyme is able to cleave them with a > or =17 nucleotide loop size at only one site under single-turnover conditions. Furthermore, an RNA cleavage reaction is detected using the immobilized deoxyribozyme on a surface plasmon resonance (SPR) sensor chip. These results show that the immobilized deoxyribozymes on a column and on an SPR sensor chip become a novel and useful tool to distinguish the RNA foldings.

Base Sequence↗
PubMed1999

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications.

Base Sequence↗
PubMed2003

Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing.

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.

Animals↗
PubMed2004

RNase P as a tool for disruption of gene expression in maize cells.

RNase P, a ribonucleoprotein responsible for the 5' maturation of precursor tRNAs (ptRNAs) in all organisms, can be enticed to cleave any target mRNA that forms a ptRNA-like structure and sequence-specific complex when bound to an RNA, termed the EGS (external guide sequence). In the present study, F3H (flavanone 3-hydroxylase), a key enzyme in the flavonoid biosynthetic pathway that participates in the formation of red-coloured anthocyanins, was used as a target for RNase P-mediated gene disruption in maize cells. Transient expression of an EGS complementary to the F3H mRNA resulted in suppression of F3H to 29% of the control, as indicated by a reduced number of anthocyanin-accumulating cells. This decrease was not observed in experiments where a disabled mutant EGS was expressed. Our results demonstrate the potential of employing plant RNase P, in the presence of an appropriate gene-specific EGS, as a tool for targeted degradation of mRNAs.

Gene Expression Regulation, Plant↗
PubMed1991

Use of bromovirus RNA3 hybrids to study template specificity in viral RNA amplification.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with genomes divided among RNAs 1, 2, and 3. RNAs 1 and 2 encode the viral RNA replication factors, which share extensive conservation with proteins encoded by the animal alphaviruses and diverse plant viruses. In barley protoplasts, CCMV RNAs 1 and 2 support high but distinguishable amplification of either BMV RNA3 (B3) or CCMV RNA3 (C3), while BMV RNAs 1 and 2 show even greater discrimination, amplifying C3 poorly relative to B3. To identify the cis-acting determinants of these template-specific and virus-specific differences in RNA3 accumulation, we constructed and tested a series of B3/C3 hybrids that exchange in turn the 5',3', and intercistronic noncoding regions, which contain all sequences required in cis for efficient B3 and C3 amplification. Despite suggestive prior in vitro results, the 3' noncoding regions were not the major determinant of the differences in amplification of B3 and C3 in vivo. Rather, 3' exchanges had relatively modest effects and did not transfer the distinctive asymmetry of amplification between B3 and C3. Intercistronic exchanges produced larger effects on RNA3 accumulation and transferred some of the polarized characteristics of the wild-type B3 and C3 behaviors. 5' exchanges revealed context-specific effects showing that the contribution of the B3 5' region to RNA3 amplification is dependent on some other B3 segment or segments. Together with previous results implicating the BMV and CCMV 1a genes in trans-acting discrimination between B3 and C3 (P. Traynor and P. Ahlquist, J. Virol. 64:69-77, 1990), these observations should help to guide studies of protein-RNA interactions governing template specificity in bromovirus RNA replication.

Cloning, Molecular↗
PubMed2003

Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors.

RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.

3T3 Cells↗