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At least 307 records · Page 17Linked to original sources
PubMed2006

Argonaute slicing is required for heterochromatic silencing and spreading.

Small interfering RNA (siRNA) guides dimethylation of histone H3 lysine-9 (H3K9me2) via the Argonaute and RNA-dependent RNA polymerase complexes, as well as base-pairing with either RNA or DNA. We show that Argonaute requires the conserved aspartate-aspartate-histidine motif for heterochromatic silencing and for ribonuclease H-like cleavage (slicing) of target messages complementary to siRNA. In the fission yeast Schizosaccharomyces pombe, heterochromatic repeats are transcribed by polymerase II. We show that H3K9me2 spreads into silent reporter genes when they are embedded within these transcripts and that spreading requires read-through transcription, as well as slicing by Argonaute. Thus, siRNA guides histone modification by basepairing interactions with RNA.

Amino Acid Motifs↗
PubMed1998

Nuclease-resistant external guide sequence-induced cleavage of target RNA by human ribonuclease P.

External guide sequences (EGSs) are short oligoribonucleotides, which are designed to bind to a given RNA target and form a precursor tRNA-like complex. This complex can be recognized by ribonuclease P (RNase P), resulting in specific cleavage of the RNA target. To explore the potential of this class of compounds as therapeutic agents and valuable tools for gene function analysis, various chemical modifications were introduced into an all-RNA EGS molecule to confer nuclease resistance. In particular, 2'-O-methyl substitutions were incorporated into the entire sequence (i.e., A-stem, D-stem, and T-stem) except the T-loop region without loss of cleavage-inducing activity. Replacement of rU (position 54) and rC (position 56) in the T-loop with their 2'-O-methyl counterparts caused pronounced decrease in activity. Moreover, phosphorothioate backbone modification of the T-loop did not provide sufficient protection against endonucleolytic attack at the ribopyrimidine residues. Systematic modification of the T-loop with a variety of modified nucleosides and the addition of a 3'-3' inverted T at the 3'-end have generated several lead EGS prototypes, which not only exhibit wild-type activity in inducing RNase P-mediated target cleavage as compared with the all-RNA control but also remain intact in human serum for more than 24 hours. These results should provide useful insights into the design and development of oligonucleotide-based EGSs as potential regulators of gene expression.

Base Sequence↗
PubMed2005

Expression of complementary RNA from chloroplast transgenes affects editing efficiency of transgene and endogenous chloroplast transcripts.

The expression of angiosperm chloroplast genes is modified by C-to-U RNA editing. The mechanism for recognition of the approximately 30 C targets of editing is not understood. There is no single consensus sequence surrounding editing sites, though sites can be grouped into small 'clusters' of two to five sites exhibiting some sequence similarity. While complementary RNA that guides nucleotides for alteration has been detected in other RNA modification systems, it is not known whether complementary RNA is involved in chloroplast editing site recognition. We investigated the effect of expressing RNA antisense to the sequences -20 to +6 surrounding the RpoB-2 C target of editing, which is a member of a cluster that includes the PsbL-1 and Rps14-1 sites. Previous experiments had shown that chloroplast rpoB transgene transcripts carrying only these 27 nt were edited in vivo at the proper C. Though transcripts carrying sequences -31 to +60 surrounding the RpoB-2 sites were edited in chloroplast transgenic plants, transcripts carrying the -31 to +62 region followed by the 27 nt complementary region were not edited at all. In contrast, a similar construct, in which the C target as well as the preceding and subsequent nucleotides were mismatched within the 27 nt region, was efficiently edited. The presence of any of the four transgenes carrying RpoB-2 sequences in sense and/or antisense orientation resulted in reduced editing at the PsbL-1 site. Chloroplast transgenic plants expressing the three different antisense RNA constructs exhibited abnormal growth and development, though plants expressing the 92 nt sense transcripts were phenotypically normal.

Chloroplasts↗
PubMed2007

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation.

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

Arabidopsis↗
PubMed1995

RNA ligase and its involvement in guide RNA/mRNA chimera formation. Evidence for a cleavage-ligation mechanism of Trypanosoma brucei mRNA editing.

RNA editing in Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. Editing is thought to be directed by guide RNAs and may proceed via a chimeric guide RNA/mRNA intermediate. We have previously shown that chimera-forming activity sediments with 19 S and 35-40 S mitochondrial ribonucleoprotein particles (RNPs). In this report we examine the involvement of RNA ligase in the production of chimeric molecules in vitro. Two adenylylated proteins of 50 and 57 kDa co-sediment on glycerol gradients with RNA ligase activity as components of the ribonucleoprotein particles. The two adenylylated proteins differ in sequence and contain AMP linked via a phosphoamide bond. Both proteins are deadenylylated by the addition of ligatable RNA substrate with the concomitant release of AMP and by the addition of pyrophosphate to yield ATP. Incubation with nonligatable RNA substrate results in an accumulation of the adenylylated RNA intermediate. These experiments identify the adenylylated proteins as RNA ligases. AMP release from the mitochondrial RNA ligase is also concomitant with chimera formation. Inhibition by nonhydrolyzable analogs indicates that both RNA ligase and chimera-forming activities require alpha-beta bond hydrolysis of ATP. Deadenylylation of the ligase inhibits chimera formation. These results strongly suggest the involvement of RNA ligase in in vitro chimera formation and support the cleavage-ligation mechanism for kinetoplastid RNA editing.

Adenosine Monophosphate↗
PubMed2002

Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein.

Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3'-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

Amino Acid Sequence↗
PubMed2003

Control of leaf morphogenesis by microRNAs.

Plants with altered microRNA metabolism have pleiotropic developmental defects, but direct evidence for microRNAs regulating specific aspects of plant morphogenesis has been lacking. In a genetic screen, we identified the JAW locus, which produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development. MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter. In addition, overexpression of wild-type and microRNA-resistant TCP variants demonstrates that mRNA cleavage is largely sufficient to restrict TCP function to its normal domain of activity. TCP genes with microRNA target sequences are found in a wide range of species, indicating that microRNA-mediated control of leaf morphogenesis is conserved in plants with very different leaf forms.

Arabidopsis↗
PubMed2000

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks.

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.

Amino Acid Sequence↗
PubMed2023

Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models.

Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.

Humans↗
PubMed2005

Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes.

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3' end of the 5' mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3'-5' exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3' overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3' overhanging Us from the bridged mRNA 5' cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.

Animals↗
PubMed2003

siRNA function in RNAi: a chemical modification analysis.

Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.

HeLa Cells↗
PubMed2006

Therapies for HIV with RNAi.

RNA interference (RNAi) is a process in which double-stranded RNA triggers the silencing of gene expression in a sequence-specific manner. In mammalian cells, approximately 21-nucleotide small interfering RNA duplexes guide cognate mRNA degradation in a sequence-specific manner by RNA-induced silencing complex. RNAi was successfully applied to inhibit different stages of HIV-1 replication. Recent viral and host cell targets for RNAi that have been used to inhibit HIV-1 are described. The problem with the delivery of RNA duplexes to the target cells and the strategies used by HIV-1 to escape inhibition by RNAi are also discussed.

Animals↗
PubMed1998

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.

Amino Acid Sequence↗
PubMed2001

Probing RNA in vivo with methylation guide small nucleolar RNAs.

Most box C/D small nucleolar RNAs (snoRNAs) direct the formation of 2'-O-methylated nucleotides in ribosomal RNA and, apparently, other RNAs present in the nucleolar complex. Sites to be modified are selected by a long (>10-nt) antisense guide sequence in the snoRNA and a distance measurement from a box D or D' element that follows the snoRNA guide sequence. Modification of the substrate occurs in the region of complementarity, at a position five nucleotides upstream from box D/D'. Methylation can be targeted to novel sites by expressing a snoRNA with a new guide sequence. In some cases methylation impairs the growth rate of the cell, indicating that a functionally important nucleotide has been altered. With a view to harnessing snoRNA-directed methylation for functional mapping, we have developed a method for constructing libraries of snoRNA genes that, in principle, can introduce methylation point mutations into any rRNA segment of interest. The strategy and procedures are described here, and preliminary results are presented that show the feasibility of using this technology to probe a region of the yeast large subunit rRNA that includes the core of the peptidyltransferase center.

Blotting, Northern↗
PubMed1997

Phylogenetic affinity of mitochondria of Euglena gracilis and kinetoplastids using cytochrome oxidase I and hsp60.

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5' and 3' untranslated transcribed sequences and lack a 3' poly[A] tail. The COI gene does not require uridine insertion/ deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [alpha-32P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3' end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences.

Animals↗
PubMed2002

Mechanism of the gBP21-mediated RNA/RNA annealing reaction: matchmaking and charge reduction.

The guide RNA-binding protein gBP21 has been characterized as a mitochondrial RNA/RNA annealing factor. The protein co-immunoprecipitates with RNA editing ribonucleoprotein complexes, which suggests that gBP21 contributes its annealing activity to the RNA editing machinery. In support of this view, gBP21 was found to accelerate the hybridization of cognate guide (g)RNA/pre-edited mRNA pairs. Here we analyze the mechanism of the gBP21-mediated RNA annealing reaction. Three possible modes of action are considered: chaperone function, matchmaker function and product stabilization. We conclude that gBP21 works as a matchmaker by binding to gRNAs as one of the two RNA annealing reactants. Three lines of evidence substantiate this model. First, gBP21 and gRNAs form a thermodynamically and kinetically stable complex in a 1 + 1 stoichiometry. Secondly, gRNA-bound gBP21 stabilizes single-stranded RNA, which can be considered the transition state in the annealing reaction. Thirdly, gBP21 has a low affinity for double-stranded RNAs, suggesting the release of the annealed reaction product after the hybridization step. In the process, up to six ionic bonds are formed between gBP21 and a gRNA, which decreases the net negative charge of the RNA. As a consequence, the electrostatic repulsion between the two annealing reactants is reduced favoring the hybridization reaction.

Adsorption↗
PubMed2026

RNA splicing and cardiovascular disease: a guide for cardiologists.

Alternative splicing (AS) is a fundamental RNA processing mechanism, which generates different RNA transcripts and consequently different protein isoforms from a single gene. This increases the diversity of proteins within an organism and can fine-tune biological processes. This review examines how cardiac-enriched RNA-binding proteins establish heart-specific splicing programs governing aspects of cardiac development, function, and disease. Developmentally, coordinated sarcomeric isoform switches underpin the foetal-to-adult transition and further isoform rewiring in ion channel and kinase genes determine electrophysiology and excitation-contraction coupling. AS contributes to the pathogenesis of several cardiomyopathies and emerging datasets suggest that pathological hypertrophy engages distinct splicing signatures compared with physiological hypertrophy. This review summarizes diagnostic and prognostic opportunities arising from bulk, long-read, and single-cell/nucleus transcriptomics, which resolve cell type-specific isoforms and disease-associated switches. Circulating RNA biomarkers (including splice ratios and circularRNAs) may signify myocardial remodelling and arrhythmic risk. Integrative approaches that link AS with proteomics and genomics improve variant interpretation, reveal previously unannotated protein isoforms, and enable tracking of disease progression and therapy response. Finally, an outline of therapeutic strategies to modulate AS in cardiovascular disease (CVD), including antisense oligonucleotides, small molecules, and genome-editing modalities (CRISPR, base, and prime editing), is provided. The major challenges that remain before splice-targeting therapeutics can be targeted to treat cardiovascular disease are highlighted. Lessons from neuromuscular indications establish clinical feasibility of splicing correction and motivate translation to cardiology. Together, mechanistic insight, biomarker development, and therapeutic innovation position RNA splicing as a tractable axis for precision cardiovascular medicine.

Humans↗