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At least 127 records · Page 7Linked to original sources
PubMed1996

Experimental verification of the secondary structures of guide RNA-pre-mRNA chimaeric molecules in Trypanosoma brucei.

RNA editing in kinetoplastid organisms is an RNA-processing reaction that adds and deletes U nucleotides at specific sites in mitochondrial pre-mRNAs. The edited sequence is specified by guide RNAs and the processing presumably occurs within a high-molecular-mass ribonucleoprotein complex containing several enzymatic activities. Although the mechanism is not currently known, potential intermediates or by-products of the editing process are chimaeric RNAs where guide (g) RNAs are covalently attached, via their non-encoded U-tail, to their cognate pre-mRNAs. We determined the secondary structures of three different ATPase 6 chimaeras of Trypanosoma brucei using a set of structure-sensitive chemical and enzymatic probes. The experiments revealed a bipartite domain structure consisting of a gRNA/pre-mRNA interaction hairpin and an independently folding mRNA stem/loop in all three RNAs. The connecting U-tail was a determinant for the length of the interaction stems with the oligo(U) nucleotides base pairing to internal gRNA sequences. The probed structures have calculated delta G27o values of -92 kJ/ mol to -134 kJ/mol, somewhat less stable than the predicted minimal free energy structures and support previously proposed models for the interaction between gRNAs and pre-mRNAs. Optical melting studies indicated additional, higher order structural features for all three molecules with four defined melting transition between 10 degrees C and 90 degrees C. A comparison of CD spectra in the absence and presence of mitochondrial protein extracts demonstrated no gross structural changes of the RNA structures induced by the association with polypeptides.

Adenosine Triphosphatases↗
PubMed2001

Cloning and characterization of two guide RNA-binding proteins from mitochondria of Crithidia fasciculata: gBP27, a novel protein, and gBP29, the orthologue of Trypanosoma brucei gBP21.

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.

Amino Acid Sequence↗
PubMed1993

The 5'-terminal region of the apocytochrome b transcript in Crithidia fasciculata is successively edited by two guide RNAs in the 3' to 5' direction.

We analyzed the chimeric guide RNA (gRNA)-mRNA molecules in Crithidia fasciculata that are predicted to transiently exist in editing of the 5'-terminal domain of apocytochrome b (CYb) mRNA, by polymerase chain reaction amplification and DNA sequencing, and obtained evidence suggesting that among the 14 editing sites numbered from 3' to 5', the sequence in the 3'-half of the sites (3' block) was specified by one guide RNA species (gRNA-I) and that in the remaining half of the sites (5' block) by the other guide RNA species (gRNA-II) and that the direction of editing in each block was 3' to 5'. The predicted transition site of editing by two gRNAs was between the first and second U residues from the 3' end within editing site 7. We found that a stretch of the edited sequence in the 3' block of mRNA could form a stable duplex with a stretch immediately upstream of the guide sequence in gRNA-II. The result leads to a successive editing model that the 3' block of pre-edited mRNA is first edited by gRNA-I, and after completion of editing, the 5' portion of gRNA-II pairs with the edited mRNA for editing of the 5' block.

Animals↗
PubMed2002

Small nucleolar RNAs: versatile trans-acting molecules of ancient evolutionary origin.

The small nucleolar RNAs (snoRNAs) are an abundant class of trans-acting RNAs that function in ribosome biogenesis in the eukaryotic nucleolus. Elegant work has revealed that most known snoRNAs guide modification of pre-ribosomal RNA (pre-rRNA) by base pairing near target sites. Other snoRNAs are involved in cleavage of pre-rRNA by mechanisms that have not yet been detailed. Moreover, our appreciation of the cellular roles of the snoRNAs is expanding with new evidence that snoRNAs also target modification of small nuclear RNAs and messenger RNAs. Many snoRNAs are produced by unorthodox modes of biogenesis including salvage from introns of pre-mRNAs. The recent discovery that homologs of snoRNAs as well as associated proteins exist in the domain Archaea indicates that the RNA-guided RNA modification system is of ancient evolutionary origin. In addition, it has become clear that the RNA component of vertebrate telomerase (an enzyme implicated in cancer and cellular senescence) is related to snoRNAs. During its evolution, vertebrate telomerase RNA appears to have co-opted a snoRNA domain that is essential for the function of telomerase RNA in vivo. The unique properties of snoRNAs are now being harnessed for basic research and therapeutic applications.

Animals↗
PubMed1999

A cis-acting A-U sequence element induces kinetoplastid U-insertions.

A 34-nucleotide A-U sequence located immediately upstream of the editing sites of the Leishmania tarentolae cytochrome b mRNA induces a mitochondrial extract to insert U nucleotides independent of guide RNA. Insertions are localized to positions immediately 5' and 3' of the A-U sequence. When placed within an unedited mammalian transcript, the A-U sequence is sufficient to induce U-insertions. The sequence has a high degree of similarity with the templating nucleotides of a cytochrome b guide RNA and with a sequence adjacent to the editing sites in ND7 mRNA, the other characterized kinetoplastid mRNA supporting guide RNA-independent U-insertions. At least one protein specifically interacts with the A-U sequence. The reaction is consistent with a mechanism proposed for guide RNA-directed editing.

Animals↗
PubMed2026

Targeting cancer-specific mutations with RNA-triggered chromatin shredding.

Genetic mutations that drive cancer often occur in tumour-suppressor proteins such as the p53 transcription factor, which is altered in 40-50% of cases1,2. However, current therapies often fail to target these mutations because the mutant proteins typically lack defined drug-binding pockets and restoring their endogenous function has proven challenging. Here we program Cas12a2, an RNA-guided CRISPR nuclease with trans-nucleolytic cleavage activity3,4, to kill cancer cells selectively by targeting cancer-specific transcripts. This approach limited cell growth by inducing trans shredding of chromatin and triggering DNA-damage responses and cell death. In contrast to existing methods, RNA-guided Cas12a2 senses cellular RNA signatures, enabling precise targeting of undruggable mutations. Transcript-activated chromatin shredding provides an innovative approach to precision disease treatments for undruggable targets.

Animals↗
PubMed2004

Chromosome healing through terminal deletions generated by de novo telomere additions in Saccharomyces cerevisiae.

Broken chromosomes healed by de novo addition of a telomere are a major class of genome rearrangements seen in Saccharomyces cerevisiae and similar to rearrangements seen in human tumors. We have analyzed the sequences of 534 independent de novo telomere additions within a 12-kb region of chromosome V. The distribution of events mirrored that of four-base sequences consisting of the GG, GT, and TG dinucleotides, suggesting that de novo telomere additions occur at short regions of homology to the telomerase guide RNA. These chromosomal sequences restrict potential registrations of the added telomere sequence. The first 11 nucleotides of the addition sequences fell into common families that included 91% of the breakpoints. The observed registrations suggest that the 3' end of the TLC1 guide RNA is involved in annealing but not as a template for synthesis. Some families of added sequences can be accounted for by one cycle of annealing and extension, whereas others require a minimum of two. The same pattern emerges for sequences added onto the most common addition sequence, indicating that de novo telomeres are added and extended by the same process. Together, these data indicate that annealing is central to telomerase registration, which limits telomere heterogeneity and resolves the problem of synthesizing Rap1 binding sites by a nonprocessive telomerase with a low-complexity guide RNA sequence.

Chromosome Aberrations↗
PubMed1999

Uridine insertion/deletion RNA editing in trypanosome mitochondria--a review.

The uridine insertion/deletion RNA editing in trypanosome mitochondria is a unique posttranscriptional RNA maturation process that involves the addition or removal of uridine residues at precise sites usually within the coding regions of mitochondrial transcripts. This process creates initiation and termination codons, corrects frameshifts and even builds entire open-reading frames from nonsense sequences. The development of several in-vitro editing assays has provided much insight into the molecular mechanism of RNA editing, which appears to involve cleavage, U addition, exonuclease trimming and ligation, essentially as proposed in the original 'enzyme cascade' model (Blum, B., Bakalara, N., Simpson, L., 1990. A model for RNA editing in kinetoplastid mitochondria: 'Guide' RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60, 189-198). However, little is known about the biochemical properties of the proteins involved and the significance and role of this process. This article is a review of recent findings on uridine-insertion/deletion editing in trypanosome mitochondria, with an emphasis on the proteins isolated and characterized that may have a role in this process.

Animals↗
PubMed1994

RNA editing: transfer of genetic information from gRNA to precursor mRNA in vitro.

RNA editing in the mitochondrion of Trypanosoma brucei extensively alters the adenosine triphosphate synthase (ATPase) subunit 6 precursor messenger RNA (pre-mRNA) by addition of 447 uridines and removal of 28 uridines. In vivo, the guide RNA gA6[14] is thought to specify the deletion of two uridines from the editing site closest to the 3' end. In this study, an in vitro system was developed that accurately removed uridines from this editing site in synthetic ATPase 6 pre-mRNA when gA6[14] and ATP were added. Mutations in both the guide RNA and the pre-mRNA editing site suggest that base-pairing interactions control the number of uridines deleted in vitro. Thus, guide RNAs are required for RNA editing and for the transfer of genetic information to pre-mRNAs.

Adenosine Triphosphatases↗
PubMed1997

The mitochondrion in dividing Leishmania tarentolae cells is symmetric and circular and becomes a single asymmetric tubule in non-dividing cells due to division of the kinetoplast portion.

Kinetoplastid protozoa have a single mitochondrion that extends throughout the cell. The disk-shaped portion of the mitochondrion adjacent to the basal body of the flagellum contains the kinetoplast DNA nucleoid body which consists of thousands of catenated minicircles and a smaller number of catenated maxicircles. The maxicircles contain structural genes and cryptogenes, rRNA genes, and a few guide RNA genes The minicircles contain the majority of the guide RNA genes. The long slender non-dividing stationary phase Leishmania tarentolae cells in culture have an asymmetric mitochondrion that consists of a single tubule extending from one edge of the kinetoplast portion. This presents a problem for cell division, in that one daughter cell will receive significantly less mitochondrial membranes than the other cell. We show in this paper that the solution to this problem is that dividing cells, which are normally shorter and rounder than stationary phase cells, possess a symmetric circular mitochondrion that has mitochondrial tubules extending from both edges of the kinetoplast which are joined in the posterior region of the cell. This implies that growth of the mitochondrion occurs after cell division, either from elongation of the longitudinal tubule towards the anterior of the cell, or from elongation of the kinetoplast portion of the mitochondrion towards the posterior region and fusion of the tubules.

Animals↗
PubMed2024

Aptazyme-directed A-to-I RNA editing.

As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-λN-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.

Animals↗
PubMed1994

RNA editing and mitochondrial genomic organization in the cryptobiid kinetoplastid protozoan Trypanoplasma borreli.

The bodonids and cryptobiids represent an early diverged sister group to the trypanosomatids among the kinetoplastid protozoa. The trypanosome type of uridine insertion-deletion RNA editing was found to occur in the cryptobiid fish parasite Trypanoplasma borreli. A pan-edited ribosomal protein, S12, and a novel 3'- and 5'-edited cytochrome b, in addition to an unedited cytochrome oxidase III gene and an apparently unedited 12S rRNA gene, were found in a 6-kb fragment of the 80- to 90-kb mitochondrial genome. The gene order differs from that in trypanosomatids, as does the organization of putative guide RNA genes; guide RNA-like molecules are transcribed from tandemly repeated 1-kb sequences organized in 200- and 170-kb molecules instead of minicircles. The presence of pan-editing in this lineage is consistent with an ancient evolutionary origin of this process.

Animals↗
PubMed2005

Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex.

RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes. Argonaute proteins, containing amino-terminal PAZ (for PIWI/Argonaute/Zwille) domains and carboxy-terminal PIWI domains, are core components of these mechanisms. Here we show the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a small interfering RNA (siRNA)-like duplex, which mimics the 5' end of a guide RNA strand bound to an overhanging target messenger RNA. The structure contains a highly conserved metal-binding site that anchors the 5' nucleotide of the guide RNA. The first base pair of the duplex is unwound, separating the 5' nucleotide of the guide from the complementary nucleotide on the target strand, which exits with the 3' overhang through a short channel. The remaining base-paired nucleotides assume an A-form helix, accommodated within a channel in the PIWI domain, which can be extended to place the scissile phosphate of the target strand adjacent to the putative slicer catalytic site. This study provides insights into mechanisms of target mRNA recognition and cleavage by an Argonaute-siRNA guide complex.

Archaeal Proteins↗
PubMed2025

Biochemical reconstitution of a type I-B CRISPR-associated transposon.

CRISPR-associated transposons (CASTs) are potential gene editing tools because of their RNA-guided DNA insertion activity. It is essential to understand the mechanisms underlying the transposition for the application of CASTs. Here, we provide protocols for the biochemical reconstitution of a type I-B CAST for RNA-guided transposition. The procedures may be applicable to other types of CASTs and facilitate the mechanism studies of various CASTs.

DNA Transposable Elements↗
PubMedDate not supplied

Using baseline CD4 cell count and plasma HIV RNA to guide the initiation of highly active antiretroviral therapy.

Conflicting evidence regarding the impact of baseline plasma HIV RNA and CD4 cell count on survival after the initiation of highly active antiretroviral therapy (HAART) in HIV-infected patients has resulted in wide variability in the expert recommendations regarding when to start therapy. Early initiation of HAART may result in avoidable toxicities and premature evolution of resistance, whereas delaying HAART may increase the risk of opportunistic infections and/or preclude a worse virological and clinical response to therapy. While there is widespread consensus that HAART can be delayed to a CD4 cell count of 0.350 x 10(9) cells/L, the range between this threshold and 0.200 x 10(9) cells/L remains controversial. Greater uncertainty surrounds the role of baseline plasma HIV RNA, with some guidelines recommending initiating HAART when this level rises above 55,000 c/mL regardless of baseline CD4 cell count. The following review examines the evidence in support of delaying the initiation of HAART to a CD4 cell count of 0.200 x 10(9) cells/L regardless of plasma HIV RNA levels and outlines supporting data from a Canadian prospective cohort study of antiretroviral naive patients treated with HAART.

Antiretroviral Therapy, Highly Active↗
PubMed2006

Hairpin RNA: a secondary structure of primary importance.

An RNA hairpin is an essential secondary structure of RNA. It can guide RNA folding, determine interactions in a ribozyme, protect messenger RNA (mRNA) from degradation, serve as a recognition motif for RNA binding proteins or act as a substrate for enzymatic reactions. In this review, we have focused on cis-acting RNA hairpins in metazoa, which regulate histone gene expression, mRNA localization and translation. We also review evolution, mechanism of action and experimental use of trans-acting microRNAs, which are coded by short RNA hairpins. Finally, we discuss the existence and effects of long RNA hairpin in animals. We show that several proteins previously recognized to play a role in a specific RNA stem-loop function in cis were also linked to RNA silencing pathways where a different type of hairpin acts in trans. Such overlaps indicate that the relationship between certain mechanisms that recognize different types of RNA hairpins is closer than previously thought.

Animals↗
PubMed2025

RNA chemistry and therapeutics.

RNA-based therapeutics have made substantial clinical advances, primarily due to the unique chemical and biological profiles of RNA molecules. As evidenced by the approval of various RNA drugs, some initial challenges related to RNA-based therapeutics, including issues associated with large-scale production, effective delivery and immunogenicity properties, are now being addressed. Extensive efforts have focused on chemically modifying RNA molecules to enhance their stability, increase protein production, extend circulation time and improve target specificity. Three RNA categories - small RNA, translatable RNA and CRISPR guide RNA - are now being extensively developed for therapeutic applications. This Review summarizes the synthetic methods applied to these three RNA categories, describes key chemical modification strategies being used to enhance their properties and highlights current therapeutic applications and future opportunities.

Humans↗