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At least 73 records · Page 4Linked to original sources
PubMed1991

Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification.

Chimeric RNA molecules were detected by polymerase chain reaction amplification of kinetoplast RNA using a 3' primer specific to mRNA and a 5' primer specific to guide RNA (gRNA), and directly by Northern analysis. Covalent linkage of the 3' oligo(U) tail of the gRNA to the mRNA occurs at editing sites. Chimeric molecules were isolated for NADH dehydrogenase subunit 7 and cytochrome oxidase subunits II and III. We propose that these molecules are intermediates in the editing process and that successive transesterifications result in the transfer of uridine residues from the gRNA 3' oligo(U) tail to an editing site, with the number of uridine residues determined by base pairing with adenine and guanine "guide" nucleotides in the gRNA.

Animals↗
PubMed1991

Cycles of progressive realignment of gRNA with mRNA in RNA editing.

We characterized numerous partially edited NADH dehydrogenase 7 and ATPase 6 cDNAs. Most of these have a stretch of incompletely edited sequence at the junction of mature and unedited sequences. The characteristics of the junctions suggest editing of sites multiple times and that editing within each junction does not proceed precisely 3' to 5'. Analyses of gRNAs and corresponding junction sequences predict a series of progressively more stable, but incompletely base-paired, interactions in the junction region. The predicted interactions suggest that the gRNA is progressively realigned with the mRNA being edited. We suggest that gRNA interactions with the mRNA result in regions of lower thermodynamic stability that are selected for editing, thus driving toward the most stable structure, the complete gRNA/mRNA duplex.

Adenosine Triphosphatases↗
PubMed1992

The polarity of editing within a multiple gRNA-mediated domain is due to formation of anchors for upstream gRNAs by downstream editing.

Seventeen kinetoplast minicircle-encoded and nine maxicircle-encoded gRNA genes have been identified. Six overlapping minicircle-encoded gRNAs mediate editing for the 5'-pan-edited MURF4 gene and two for the 5'-edited COIII gene. The pan-edited RPS12 mRNA is edited by seven minicircle-encoded gRNAs and one maxicircle-encoded gRNA. The 3'-most gRNA in each domain forms an anchor with unedited mRNA, whereas upstream gRNAs form anchors only with edited mRNA, thereby explaining the observed 3' to 5' polarity of editing within an editing domain. We suggest that a role of G-U base pairs is to allow breathing of the edited mRNA-gRNA hybrid and formation of the upstream anchor hybrid.

Animals↗
PubMed2024

Self-quenched tRNA reporters for imaging tRNA-derived RNA biogenesis.

tRNA-derived small RNAs (tDRs) are an emerging class of small non-coding RNAs that play crucial roles in various cellular processes. However, there is a paucity of data on their sub-cellular localization due to a lack of tools and reagents to image tDRs. Imaging tDRs remains challenging due to the similar sequences between tDR and its parent tRNA. Here, we describe an innovative tool for studying the formation and localization of tDRs in various biological processes using a self-quenched tDR biogenesis reporter. This method utilizes a full-length tRNA molecule conjugated with both fluorescence and quencher groups at 5'- and 3'- ends. In its intact state, the fluorescence is quenched. Upon cleavage by specific ribonucleases and strand separation, the fluorescence becomes detectable, allowing real-time imaging of tDR biogenesis. This protocol details the design, synthesis, and application of this reporter, including transfection procedures and imaging techniques. The method offers a powerful approach for investigating tDR dynamics in living cells, providing insights into their roles in cellular processes and stress responses.

RNA, Transfer↗
PubMed2006

An E. coli lon mutant conferring partial resistance to colicin may reveal a novel role in regulating proteins involved in the translocation of colicin.

Initially, we found that a lon mutant confers partial resistance against colicin. The results of Western blotting detected a decrease in the protein expression levels of BtuB and OmpF involved in colicin translocation in the lon mutant. Moreover, 2-D gel analysis revealed that the expression level of some scavenger proteins marks the lon mutant as being in a situation similar to oxidative stress. OxyRS and SoxRS are the two major response regulators for oxidative stress. Our RT-PCR analysis revealed an elevation of expression of the oxyS gene in the lon mutant. An immunoblot assay further confirmed that overexpression of oxyS RNA can negatively control on the expression of BtuB protein. Probably the BtuB is negatively regulated by a global regulator, oxyS, induced during oxidative stress.

Bacterial Outer Membrane Proteins↗
PubMed2004

Effective inhibition of HIV-1 replication in cultured cells by external guide sequences and ribonuclease P.

We examined the suppressive effect of HIV-1 RNA gene cleavage on HIV-1 expression, using the catalytic RNA subunit RNase P and the 3'-half tRNA(Try) [external guide sequence (EGS)] in cultured cells. HIV-1 expression was inhibited by the tRNA(met)-EGS-U5 and U6-EGS-U5 from the tRNA(met) and U6 promoters, respectively. There was no difference in the inhibitory effects on HIV-1 expression between the tRNA(met) and U6 promoters.

Anti-HIV Agents↗
PubMed2004

Guide RNAs with 5' caps and novel box C/D snoRNA-like domains for modification of snRNAs in metazoa.

BACKGROUND: Spliceosomal snRNAs and ribosomal RNAs in metazoans contain numerous modified residues that are functionally important. The most common modifications are site-specific 2'-O-methylation and pseudouridylation, both directed by small ribonucleoprotein particles. Each particle is composed of a short guide RNA and a set of several proteins. All previously characterized modification guide RNAs in metazoa are encoded in and processed from introns. RESULTS: We have identified and characterized three novel guide RNAs for conserved 2'-O-methylation of U2, U4, and U12 snRNAs. Two guides, termed mgU2-25/61 and mgU12-22/U4-8, appear to be independently transcribed as judged by the presence of methylated guanosine caps at their 5' ends and upstream promoters similar to those of telomerase RNA. These guide RNAs are each composed of a canonical box C/D snoRNA and a novel box C/D snoRNA-like domain, where the C'/D' motif, rather than C/D, can be folded into a conserved kink-turn structure. The snoRNA-like domains are predicted to direct 2'-O-methylation of invariant G residues that occupy analogous positions in the U2 and U12 snRNA secondary structures. A third guide, mgU2-19/30 RNA, is composed of two canonical box C/D snoRNA domains encoded within a single intron. CONCLUSIONS: This is the first description in metazoan cells of 5'-capped modification guide RNAs that appear to be independently transcribed. Since plant, yeast, and protozoan guide RNAs are mostly independently transcribed, the identification of such RNAs argues that ancestral metazoans possessed independently transcribed guide RNAs and only later, during the evolution of metazoan organisms, did the guide RNA genes shift to introns.

Animals↗
PubMed2003

Functional requirement for symmetric assembly of archaeal box C/D small ribonucleoprotein particles.

Box C/D small ribonucleoprotein particles (sRNPs) are archaeal homologs of small nucleolar ribonucleoprotein particles (snoRNPs) in eukaryotes that are responsible for site specific 2'-O-methylation of ribosomal and transfer RNAs. The function of box C/D sRNPs is characterized by step-wise assembly of three core proteins around a box C/D RNA that include fibrillarin, Nop5p, and L7Ae. The most distinct structural feature in all box C/D RNAs is the presence of two conserved box C/D motifs accompanied by often a single, and sometimes two, antisense elements located immediately upstream of either the D or D' box. Despite this asymmetric distribution of antisense elements, the bipartite feature of the box C/D motifs appears to be in pleasing agreement with a recently reported three-dimensional structure of the core protein complex between fibrillarin and Nop5p. This investigates functional implications of the symmetric features both in box C/D RNAs and in the fibrillarin-Nop5p complex. Site-directed mutagenesis was employed to generate box C/D RNAs lacking one of the two box C/D motifs and a mutant fibrillarin-Nop5p complex deficient in self-association. The ability of the mutated components to assemble and to direct methyl transfer reactions was assessed by gel mobility-shift, analytical ultracentrifugation, and in vitro catalysis studies. The results presented here suggest that, while a box C/D sRNP is capable of asymmetrical assembly, the symmetries in both the box C/D RNA and in the fibrillarin-Nop5p complex are required for efficient catalysis. These findings underscore the importance of functional assembly in methyl transfer reactions.

Archaeal Proteins↗
PubMed2006

Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling.

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.

Amino Acid Sequence↗
PubMed2026

A muskrat tissue atlas of small non-coding RNAs and their regulatory roles in muskrat musk secretion.

Muskrat musk, secreted by the male muskrat scent gland during the secretion period, is a valuable natural product with promising pharmacological activities. Its synthesis is regulated not only by coding genes but also by small non-coding RNAs (sncRNAs). However, a comprehensive tissue atlas of sncRNAs in muskrats has been lacking. To address this issue, we systematically profiled the expression levels of 6 sncRNA categories (piRNA [PIWI-interacting RNA], miRNA [microRNA], snoRNA [small nucleolar RNA], snRNA [small nuclear RNA], tRNA [transfer RNA], and other RNAs) across 13 tissues of the muskrat via deep sequencing. We identified 23,957 sncRNAs (∼ 65%) exhibiting tissue-specific expression patterns throughout the body. The results show that miRNA is the main contributor to tissue specificity. Eleven tissues showed a significantly higher number of miRNA-5p arm expressions compared to the 3p arm, although the brain was an exception. Quantitative results imply that miR-477-3p/5p and miR-794-3p are potentially involved in the regulation of muskrat musk synthesis and secretion in the muskrat scent glands. Their target genes were enriched in pathways related to energy supply, lipid metabolism, and cyclic morphological changes of the scent gland. Furthermore, we constructed a set of co-expressed miRNAs based on the hormone-dominated "brain-testis-scent gland" axis and the energy metabolism-dominated "brain-liver-scent gland" axis. These results provide the most comprehensive description to date of tissue-specific and ubiquitous sncRNAs in individual muskrat tissues. We anticipate that these data will enhance the understanding of the molecular regulation underlying muskrat musk synthesis and secretion.

Animals↗
PubMed2002

Ribosomal RNA pseudouridines and pseudouridine synthases.

Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.

Animals↗
PubMed2002

The expanding snoRNA world.

In eukaryotes, the site-specific formation of the two prevalent types of rRNA modified nucleotides, 2'-O-methylated nucleotides and pseudouridines, is directed by two large families of snoRNAs. These are termed box C/D and H/ACA snoRNAs, respectively, and exert their function through the formation of a canonical guide RNA duplex at the modification site. In each family, one snoRNA acts as a guide for one, or at most two modifications, through a single, or a pair of appropriate antisense elements. The two guide families now appear much larger than anticipated and their role not restricted to ribosome synthesis only. This is reflected by the recent detection of guides that can target other cellular RNAs, including snRNAs, tRNAs and possibly even mRNAs, and by the identification of scores of tissue-specific specimens in mammals. Recent characterization of homologs of eukaryotic modification guide snoRNAs in Archaea reveals the ancient origin of these non-coding RNA families and offers new perspectives as to their range of function.

Animals↗
PubMed2002

Archaeal guide RNAs function in rRNA modification in the eukaryotic nucleus.

In eukaryotes, many Box C/D small nucleolar RNAs base pair with ribosomal RNA through short complementary guide sequences, thereby marking up to 100 individual nucleotides of ribosomal RNA for 2'-O-methylation. Function of the eukaryotic Box C/D RNAs depends upon interaction with at least six known proteins. Box C/D RNAs are not known to exist in Bacteria but were recently identified in Archaea by biochemical analysis and computational genomic screens and have likely evolved independently in Archaea and Eukarya for more than 2000 million years. We have microinjected Box C/D RNAs from Pyrococcus furiosus, a hyperthermophilic archaeon, into the nuclei of oocytes from the aquatic frog Xenopus laevis. Our results show that Box C/D RNAs derived from this prokaryote are retained in the nucleus, localize to nucleoli, and interact with the X. laevis Box C/D RNA binding proteins fibrillarin, Nop56, and Nop58. Furthermore, we have demonstrated the ability of archaeal Box C/D RNAs to direct site-specific 2'-O-methylation of ribosomal RNA. Our studies have revealed the remarkable ability of archaeal Box C/D RNAs to assemble into functional RNA-protein complexes in the eukaryotic nucleus.

Active Transport, Cell Nucleus↗
PubMed1997

RNA processing: pocket guides to ribosomal RNA.

The functional role of a recently identified class of small nucleolar (sno) RNAs has been elucidated: the 'box H/ACA' snoRNAs act as guide RNAs, specifying the position of evolutionarily conserved pseudouridines in ribosomal (r)RNA via an rRNA-snoRNA base-pairing interaction that forms a 'pseudouridine pocket'.

Base Composition↗
PubMed1998

Birth of the snoRNPs: the evolution of the modification-guide snoRNAs.

Bacteria and eukaryotes adopt very different strategies to modify their rRNAs. Most sites of eukaryotic rRNA modification are selected by guide small nucleolar RNAs (snoRNAs), while bacteria rely on numerous site-specific modification enzymes. This raises a 'chicken and egg' dilemma: how could a system of modification that requires a large number of snoRNA cofactors have developed? Did it arise in a de novo fashion, or evolve from a pre-existing protein-based system? The rRNA sequences are well conserved in evolution, but the pattern of modification is only moderately conserved, and many more sites are modified in eukaryotes than in bacteria; why is this so? We propose a model for the origins of the modification-guide snoRNAs that attempts to answer these questions.

Cell Nucleolus↗
PubMed2002

The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs.

The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation. Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells. Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU. We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins.

Amino Acid Sequence↗
PubMed2002

A BLAST from the past: ancient origin of human Sm proteins.

In this issue of Molecular Cell, Zhang et al. and Møller et al. independently report studies of the E. coli Hfq protein, revealing significant sequence similarities with human Sm proteins. Their findings suggest that Hfq, and the Sm and Sm-like (Lsm) proteins, may function in stabilizing interactions between RNA molecules.

Autoantigens↗