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At least 397 records · Page 22Linked to original sources
PubMed2001

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

Posttranscriptional editing of trypanosome mitochondrial messenger RNA is directed by small guide RNAs (gRNAs). Using crosslinking techniques, we have previously shown that the gRNA base pairs to the mRNA via a 5' anchor, whereas its 3' U-tail interacts with upstream purine-rich mRNA sequences. The incorporation of crosslinking data into RNA folding programs produced similar structure predictions for all gRNA/mRNA pairs examined. This suggests that gRNA/mRNA pairs can form common secondary structure motifs that may be important for recognition by the editing complex. In this study, the structure of CYb mRNA crosslinked to gCYb-558 was examined using solution-probing techniques. The mRNA/gRNA crosslinked molecules are efficient substrates for gRNA-directed cleavage. In addition, when the cleavage assay is performed in the presence or absence of additional UTP, the activities of both the U-specific exonuclease and terminal uridylyl transferase (tutase) can be detected. These results indicate that a partial editing complex can assemble and function on these substrates suggesting that the crosslink captured the molecules in a biologically relevant interaction. The structure probing data directly show that the U-tail protects several mRNA bases predicted to be involved in the U-tail-mRNA duplex. In combination with our previous studies, these new data provide additional support for the predicted secondary structure of interacting gRNA/mRNA pairs.

Animals↗
PubMed1992

In vitro guide RNA/mRNA chimaera formation in Trypanosoma brucei RNA editing.

The post-transcriptional processing of various mitochondrial transcripts in kinetoplastids, kRNA editing, adds and removes uridines, producing mature messenger RNAs. This editing seems to be directed by 'guide' RNAs (gRNAs) which are complementary to portions of the mature message. The editing mechanism has been proposed to entail transesterification. Detection of chimaeric gRNA-mRNA molecules, intermediates predicted by transesterification, support this model. We report here the in vitro formation of such chimaeras where endogenous gRNAs are covalently linked to added synthetic mRNA. Addition of gel-purified gRNAs to the standard reaction mix increases chimaera formation. This increase is not observed when the gRNA 3'-hydroxyl group is chemically modified, identifying this terminal hydroxyl as the reactive group. These results provide the first experimental evidence for an in vitro RNA editing event and support the involvement of transesterification as a chemical mechanism.

Animals↗
PubMed2000

Optical spectroscopic study of the effects of a single deoxyribose substitution in a ribose backbone: implications in RNA-RNA interaction.

The 2'-OH group in the ribose sugars of an RNA molecule plays an important role in guiding tertiary interactions that stabilize different RNA structural motifs. Deoxyribose, or 2'-OH by 2'-H, substitution in both the single-stranded and the duplex part of an RNA backbone has been routinely used to evaluate what role the 2'-OH plays in different tertiary interactions that guide an RNA-RNA contact. A deoxyribose substitution not only has the effect of removing a hydrogen bond donating group, but also introduces a sugar moiety with a preference for C2'-endo pucker in a backbone of predominantly C3'-endo sugars. This study evaluates the effects of a single deoxyribose substitution in both single-stranded and double-helical forms of RNA oligomers. A single-stranded, nonrepetitive 7-mer oligoribonucleotide (7-mer RNA) and four different variants having the same base sequence but with a single deoxyribose sugar at different positions in the strands have been studied by ultraviolet (UV) absorption, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. Duplexes were formed by association with the complementary strand of the 7-mer RNA. The results show that both RNA and DNA single strands have preorganized conformations with spectral properties resembling those of A- and B-form helices, respectively, with RNA being more heterogeneous than its DNA counterpart. A single deoxyribose substitution perturbs the structure of the RNA backbone, with the effect being more pronounced in the single-stranded than in the duplex structure. The perturbation depends on the position of the 2'-H substitution in the strand.

Base Composition↗
PubMed2006

Crystal structure of an H/ACA box ribonucleoprotein particle.

H/ACA ribonucleoprotein particles (RNPs) are a family of RNA pseudouridine synthases that specify modification sites through guide RNAs. They also participate in eukaryotic ribosomal RNA processing and are a component of vertebrate telomerases. Here we report the crystal structure, at 2.3 A resolution, of an entire archaeal H/ACA RNP consisting of proteins Cbf5, Nop10, Gar1 and L7ae, and a single-hairpin H/ACA RNA, revealing a modular organization of the complex. The RNA upper stem is bound to a composite surface formed by L7ae, Nop10 and Cbf5, and the RNA lower stem and ACA signature motif are bound to the PUA domain of Cbf5, thereby positioning middle guide sequences so that they are primed to pair with substrate RNA. Furthermore, Gar1 may regulate substrate loading and release. The structure rationalizes the consensus structure of H/ACA RNAs, suggests a functional role of each protein, and provides a framework for understanding the mechanism of RNA-guided pseudouridylation, as well as various cellular functions of H/ACA RNP.

Archaeal Proteins↗
PubMed2006

Substrate determinants for RNA editing and editing complex interactions at a site for full-round U insertion.

Multisubunit RNA editing complexes catalyze uridylate insertion/deletion RNA editing directed by complementary guide RNAs (gRNAs). Editing in trypanosome mitochondria is transcript-specific and developmentally controlled, but the molecular mechanisms of substrate specificity remain unknown. Here we used a minimal A6 pre-mRNA/gRNA substrate to define functional determinants for full-round insertion and editing complex interactions at the editing site 2 (ES2). Editing begins with pre-mRNA cleavage within an internal loop flanked by upstream and downstream duplexes with gRNA. We found that substrate recognition around the internal loop is sequence-independent and that completely artificial duplexes spanning a single helical turn are functional. Furthermore, after our report of cross-linking interactions at the deletion ES1 (35), we show for the first time editing complex contacts at an insertion ES. Our studies using site-specific ribose 2' substitutions defined 2'-hydroxyls within the (a) gRNA loop region and (b) flanking helixes that markedly stimulate both pre-mRNA cleavage and editing complex interactions at ES2. Modification of the downstream helix affected scissile bond specificity. Notably, a single 2'-hydroxyl at ES2 is essential for cleavage but dispensable for editing complex cross-linking. This study provides new insights on substrate recognition during full-round editing, including the relevance of secondary structure and the first functional association of specific (pre-mRNA and gRNA) riboses with both endonuclease cleavage and cross-linking activities of editing complexes at an ES. Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript.

Animals↗
PubMed2005

Short interfering RNAs as a tool for cancer gene therapy.

There are mainly two types of short RNAs that target complementary messengers in animals: small interfering RNAs and micro-RNAs. Both are produced by the cleavage of double-stranded RNA precursors by Dicer, a member of the Rnase III family of double-stranded specific endonucleases, and both guide the RNA-induced silencing complex to cleave specifically RNAs sharing sequence identity with them. In designing a particular RNA interference (RNAi), it is important to identify the sense/antisense combination that provides the most potent suppression of the target mRNA, and several rules have been established to give >90% gene expression inhibition. RNAi technology can be directed against cancer using a variety of strategies. These include the inhibition of overexpressed oncogenes, blocking cell division by interfering with cyclin E and related genes or promoting apoptosis by suppressing antiapoptotic genes. RNAi against multidrug resistance genes or chemoresistance targets may also provide useful cancer treatments. Studies investigating these approaches in preclinical models are also reviewed.

Apoptosis↗
PubMed1999

Nucleolar factors direct the 2'-O-ribose methylation and pseudouridylation of U6 spliceosomal RNA.

The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place. In this study, we demonstrate that the nucleolus contains all the trans-acting factors that are responsible for the accurate and efficient synthesis of the eight 2'-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA. Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus. For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2'-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified. This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2'-O-methylation of the U6 spliceosomal RNA (K. T. Tycowski, Z.-H. You, P. J. Graham, and J. A. Steitz, Mol. Cell 2:629-638, 1998). We demonstrate that a novel 2'-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2'-O-methylation small nucleolar guide RNA. We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2'-O-methylated and pseudouridylated. These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.

Base Sequence↗
PubMed2003

A tale of two TUTases.

The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3' terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3' oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purification-tagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3' end of a single-stranded RNA, thereby confirming that RET2 is a 3' TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro U-insertion editing and has no effect on the length of the 3' oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3' end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.

Animals↗
PubMed2005

A member of the polymerase beta nucleotidyltransferase superfamily is required for RNA interference in C. elegans.

RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression . Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins . Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility . Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3. Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase beta nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2'-5' oligoadenylate synthetase (OAS), and yeast Trf4p . These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.

Amino Acid Sequence↗
PubMed2002

Biogenesis of small nucleolar ribonucleoproteins.

Eukaryotic cells contain a very complex population of small nucleolar RNAs. They function, as small nucleolar ribonucleoproteins, in pre-ribosomal RNA processing reactions, and also guide methylation and pseudouridylation of ribosomal RNA, spliceosomal small nuclear RNAs, and possibly other cellular RNAs. Synthesis of small nucleolar RNAs frequently follows unusual strategies. Some newly discovered brain-specific small nucleolar RNAs of unknown function are encoded in introns of tandemly repeated units, expression of which is paternally imprinted. Recent studies of the protein components and factors participating in small nucleolar ribonucleoprotein assembly have revealed interesting connections with other classes of cellular ribonucleoproteins such as spliceosomal small nuclear ribonucleoproteins and telomerase. Cajal bodies emerge as nuclear structures important for the biogenesis and function of small nucleolar ribonucleoproteins.

Animals↗
PubMed1999

Mammalian Hsp70 and Hsp110 proteins bind to RNA motifs involved in mRNA stability.

In this study, in vitro RNA binding by members of the mammalian 70-kDa heat shock protein (Hsp) family was examined. We show that Hsp/Hsc70 and Hsp110 proteins preferentially bound AU-rich RNA in vitro. Inhibition of RNA binding by ATP suggested the involvement of the N-terminal ATP-binding domain. By using deletion mutants of Hsp110 protein, a diverged Hsp70 family member, RNA binding was localized to the N-terminal ATP-binding domain of the molecule. The C-terminal peptide-binding domain did not bind RNA, but its engagement by a peptide substrate abrogated RNA binding by the N terminus of the protein. Interestingly, removal of the C-terminal alpha-helical structure or the alpha-loop domain unique to Hsp110 immediately downstream of the peptide-binding domain, but not both, resulted in considerably increased RNA binding as compared with the wild type protein. Finally, a 70-kDa activity was immunoprecipitated from RNA-protein complexes formed in vitro between cytoplasmic proteins of human lymphocytes and AU-rich RNA. These findings support the idea that certain heat shock proteins may act as RNA-binding entities in vivo to guide the appropriate folding of RNA substrates for subsequent regulatory processes such as mRNA degradation and/or translation.

3' Untranslated Regions↗
PubMed2026

RNA structures regulate norovirus life cycle and enable rational attenuation in vivo.

Viral genomes encode regulatory RNA structures that orchestrate key steps of viral replication and gene expression. Although these structures are increasingly recognized as critical regulators of viral function, their systematic characterization in an infection context and roles in regulating viral fitness and immune recognition in vivo remain limited. Here, we systematically map and functionally interrogate structured RNA elements across the murine norovirus genome using orthogonal in-cell chemical probing, revealing conserved motifs that regulate viral function. Targeted disruption of specific structural elements reduces viral replication in cell culture, modulates translation in cis, and decreases viral RNA levels in animal infection models. These findings enabled the rational design of a genetically stable, attenuated virus that elicits protective immunity and limits viral replication upon secondary challenge. Together, this work uncovers essential roles for RNA structure in norovirus biology and establishes a generalizable framework for RNA structure-guided design of antiviral vaccines and therapeutics.

RNA structure↗
PubMed2005

Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation.

The mRNA-cleavage step of RNA interference is mediated by an endonuclease, Argonaute2 (Ago2), within the RNA-induced silencing complex (RISC). Ago2 uses one strand of the small interfering (si) RNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone at a specific site measured from the guide strand's 5' end. Here, we show that both strands of siRNA get loaded onto Ago2 protein in Drosophila S2 cell extracts. The anti-guide strand behaves as a RISC substrate and is cleaved by Ago2. This cleavage event is important for the removal of the anti-guide strand from Ago2 protein and activation of RISC.

Adenosine Triphosphate↗
PubMed1997

Nonhomologous RNA recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure.

Extensive nonhomologous recombinations occur between the 5' and 3' fragments of a replicable RNA in a cell-free system composed of pure Qbeta phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3' hydroxyl group of the 5' fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3' hydroxyl on phosphoester bonds within the 3' fragment.

Base Sequence↗
PubMed2003

Dyskinetoplastic Trypanosoma brucei contains functional editing complexes.

Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex. Trypanosoma evansi AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of Trypanosoma brucei EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.

Animals↗
PubMed2005

Determinants of transcription initiation by archaeal RNA polymerase.

Transcription in Archaea is catalyzed by an RNA polymerase that is most similar to eukaryotic RNA polymerases both in subunit composition and in transcription initiation factor requirements. Recent studies on archaeal transcription in diverse members of this domain have contributed new details concerning the functions of promoters and transcription factors in guiding initiation by RNA polymerase, and phylogenetic arguments have allowed modeling of archaeal transcription initiation complexes by comparison with recently described models of eukaryotic and bacterial transcription initiation complexes. Important new advances in reconstitution of archaeal transcription complexes from fully recombinant components is permitting testing of hypotheses derived from and informed by these structural models, and will help bring the study of archaeal transcription to the levels of understanding currently enjoyed by bacterial and eukaryotic RNA polymerase II transcription.

Amino Acid Sequence↗
PubMed2002

RNA folding in vivo.

RNA folding in vivo is influenced by the cellular environment, the vectorial nature of transcription and translation, trans-acting factors and ion homeostasis. Specific RNA-binding proteins promote RNA folding by stabilizing the native structure or by guiding folding. In contrast, RNA chaperones, which are believed to interact nonspecifically with RNA, were proposed to resolve misfolded RNA structures and to promote intermolecular RNA-RNA annealing. Small trans-acting noncoding RNAs are thought to modulate mRNA structures, thereby regulating gene expression. So far, there is some evidence that in vitro and invivo RNA folding pathways share basic features. However, it is unclear whether the rules deduced from in vitro folding experiments generally apply to invivo conditions.

3' Flanking Region↗
PubMed2003

From the nucleus toward the cell periphery: a guided tour for mRNAs.

RNA processing, directed transport along cytoskeletal tracks, and site-specific translation of mRNA at the cell periphery are considered discrete steps in the generation of microfilament-membrane adhesion complexes. A recently identified member of the heterogeneous nuclear ribonucleoprotein family, raver1, may couple these steps and contribute to the assembly and maintenance of these structures.

Animals↗