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At least 199 records · Page 11Linked to original sources
PubMed2004

Genome-wide searching for pseudouridylation guide snoRNAs: analysis of the Saccharomyces cerevisiae genome.

One of the largest families of small RNAs in eukaryotes is the H/ACA small nucleolar RNAs (snoRNAs), most of which guide RNA pseudouridine formation. So far, an effective computational method specifically for identifying H/ACA snoRNA gene sequences has not been established. We have developed snoGPS, a program for computationally screening genomic sequences for H/ACA guide snoRNAs. The program implements a deterministic screening algorithm combined with a probabilistic model to score gene candidates. We report here the results of testing snoGPS on the budding yeast Saccharomyces cerevisiae. Six candidate snoRNAs were verified as novel RNA transcripts, and five of these were verified as guides for pseudouridine formation at specific sites in ribosomal RNA. We also predicted 14 new base-pairings between snoRNAs and known pseudouridine sites in S.cerevisiae rRNA, 12 of which were verified by gene disruption and loss of the cognate pseudouridine site. Our findings include the first prediction and verification of snoRNAs that guide pseudouridine modification at more than two sites. With this work, 41 of the 44 known pseudouridine modifications in S.cerevisiae rRNA have been linked with a verified snoRNA, providing the most complete accounting of the H/ACA snoRNAs that guide pseudouridylation in any species.

Algorithms↗
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↗
PubMed1999

The mitochondrial RNA ligase from Leishmania tarentolae can join RNA molecules bridged by a complementary RNA.

A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K(m) of 25 +/- 0.75 nM and a V(max) of 1.0 x 10(-4) +/- 2.4 x 10(-4) nmol/min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.

Adenosine Triphosphate↗
PubMed2006

Argonaute: A scaffold for the function of short regulatory RNAs.

Argonaute is the central protein component of RNA-silencing mechanisms. It provides the platform for target-mRNA recognition by short regulatory guide RNA strands and the Slicer catalytic activity for mRNA cleavage in RNA interference. Multiple Argonaute sub-families can be identified phylogenetically yet, despite this diversity, molecular and sequence analyses show that Argonaute proteins share common molecular properties and the capacity to function through a common mechanism. Recently, the members of the Piwi sub-family have been shown to interact with new classes of short regulatory RNAs, Piwi-interacting RNAs (piRNAs) and repeat-associated small interfering RNAs (rasiRNAs), which has implications for developmental processes and introduces a new dimension to the field of RNA silencing.

Binding Sites↗
PubMed2002

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.

Animals↗
PubMed2026

Rational and computation-assisted engineering of a compact and efficient CRISPR-Cas12f genome editor.

The CRISPR-Cas12f system is an ultracompact genome-editing platform, yet only a few orthologs exhibit robust activity in mammalian cells. Here, we systematically screened 23 Cas12f orthologs and identified two active nucleases, PspCas12f1 and TcCas12f1, capable of genome editing in human cells. Single guide RNA (sgRNA) scaffold optimization enhanced the basal activity of PspCas12f1. To further improve its performance, we combined structure-guided rational design with protein language model-assisted filtering. Candidate mutations predicted by SaProt were further screened based on structural proximity to the DNA-binding interface and electrostatic compatibility. This integrative strategy identified Q100R and E293R, whose combination yielded the optimized variant enPspCas12f1. enPspCas12f1 achieved genome-editing efficiencies comparable to SpCas9 across multiple endogenous loci while maintaining high specificity. Collectively, our results demonstrate that integrating protein language model-assisted filtering with structure-guided rational design provides an effective strategy for engineering PspCas12f1 and may facilitate the optimization of additional compact CRISPR nucleases.

CRISPR-Cas12f↗
PubMed2005

RNAi: the nuts and bolts of the RISC machine.

Research on RNA interference and microRNA pathways continues to accelerate. Combinations of biochemical, genetic, and structural approaches are providing new insights into the mechanism by which small, approximately 21 nt long RNAs find their way into the RISC effector complexes and how the RISCs execute their mission of RNA-guided posttranscriptional gene silencing.

Animals↗
PubMed2006

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins.

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.

Amino Acid Sequence↗
PubMed2005

Maintaining a conserved methylation in plant and insect U2 snRNA through compensatory mutation by nucleotide insertion.

The extensive post-transcriptional modification of U2 snRNA is required for spliceosome assembly and pre-mRNA splicing in vertebrates. However, the rare modification of U2 snRNA in yeast implies a different mechanism for regulating spliceosome biogenesis in single-celled eukaryotes. To understand the evolutionary pattern of U2 snRNA methylation, we determined for the first time, the 2'-O-methylations of U2 snRNA in Oryza sativa, Arabidopsis thaliana and Drosophila melanogaster, and revealed two methylations which are conserved in a crucial region of U2 snRNA in plants. Interestingly, one of the methylations, U2-Cm29 is also methylated in D. melanogaster, but not in vertebrates. According to the methylation of U2-C29, computational analysis of databases identified three canonical box C/D snoRNAs, named OsmgU2-29, AtmgU2-29 and DmmgU2-28, as small methylation guides of U2 snRNA from O. sativa, A. thaliana and D. melanogaster, respectively. Although very divergent in their sequence, the three snoRNAs exhibit in common an 11 nucleotide-long sequence complementarity to corresponding U2 snRNA, implying a functional constraint on the modification during evolution. Interestingly, a nucleotide is found to be inserted both in U2 snRNA and DmmgU2-28 and maintains a perfect match of duplex specifying the methylation of C28 in Drosophila U2 snRNA. This is the first time a new model is being provided for compensatory mutations between a small guide RNA and its target by nucleotide insertion, instead of the known nucleotide substitution. In contrast to small Cajal body-specific RNAs (scaRNAs), the snoRNAs are similar to the reported singlet guide RNAs and are known to localize in nucleolus.

Animals↗
PubMed2004

RNA silencing: a conserved antiviral immunity of plants and animals.

RNA silencing is a novel RNA-guided gene regulatory mechanism operational in a wide range of eukaryotic organisms from fission yeast, plants, to mammals. This article reviews the recent progress on aspects of RNA silencing that are related to its biological function as a conserved antiviral immunity of plants and animals, and highlights features of this novel antiviral response in invertebrate animals as compared to the known innate and adaptive immunities. Finally, we discuss evidence that suggests a natural antiviral role for RNA silencing in vertebrates as well as experimental approaches that may facilitate the identification of first mammalian viral suppressors of RNA silencing.

Animals↗
PubMed1998

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780-786).

Alternative Splicing↗
PubMed1992

[Advances in molecular parasitology].

Recent studies on molecular biology of parasites revealed novel mechanisms for the control of expression of parasitic genes. In trypanosomatid protozoa, unusual characteristics of gene regulation, including polycistronic transcription, trans-RNA splicing and RNA editing have been discovered. This review describes the recent progress of RNA editing in trypanosomes, and trans-splicing in both trypanosomes and nematodes. Studies of the editing of mRNAs in trypanosomatid mitochondria indicate the involvement of small guide RNA molecules, which are encoded in the minicircles and/or maxicircles of kinetoplast DNA. A proposed transesterification model for RNA editing suggests an analogous reaction between RNA editing and RNA splicing. In addition, the development of stable transfection system and success of homologous gene targeting in Trypanosoma and Leishmania are reported.

Animals↗
PubMed1994

RNA editing in trypanosomes.

The nucleotide sequence of mitochondrial pre-mRNAs in trypanosomes is posttranscriptionally edited by the insertion and deletion of uridylate (U) residues. In some RNAs editing is limited to small sections but in African trypanosomes, such as Trypanosoma brucei, 9 of the 18 known mitochondrial mRNAs are created by massive editing which can produce more than 50% of the coding sequence. In all cases, however, RNA editing is a key event in gene expression during which translatable RNAs are generated. The information for the editing process and possibly also the inserted Us are provided by small guide RNAs, which are encoded in both the maxicircle and minicircle components of the trypanosome mitochondrial DNA. Current models of editing are largely based on the characteristics of partially edited RNAs and on the occurrence in vivo and the possibility of synthesis in vitro of chimeric molecules in which a guide RNA is covalently linked through its 3' oligo(U) tail to an editing site in pre-mRNA. In this paper, I will review the research in this rapidly growing field and illustrate how different interpretations of the available data can lead to different views of the mechanism and the biochemistry of the editing process.

Animals↗
PubMed1982

Making ends meet: a model for RNA splicing in fungal mitochondria.

On the basis of available nucleotide sequence and genetic data; we present a model for RNA splicing in fungal mitochondria. Seven intron RNAs of two fungal species can form identical secondary structures, involving four conserved sequences, which bring the ends of each intron together and allow an internal guide RNA sequence to pair with exon bases adjacent to the splice junctions. The splicing sites are thus aligned precisely within a conserved structure, which we suggest could present specific recognition signals to the proteins that catalyse the splicing reaction.

Aspergillus nidulans↗
PubMed2005

Ribo-gnome: the big world of small RNAs.

Small RNA guides--microRNAs, small interfering RNAs, and repeat-associated small interfering RNAs, 21 to 30 nucleotides in length--shape diverse cellular pathways, from chromosome architecture to stem cell maintenance. Fifteen years after the discovery of RNA silencing, we are only just beginning to understand the depth and complexity of how these RNAs regulate gene expression and to consider their role in shaping the evolutionary history of higher eukaryotes.

Animals↗
PubMed1996

Mitochondrial RNA editing is sequence specific and independent of transcript abundance in Sorghum bicolor.

The DNA sequence which encodes the amino-terminal extension to the conserved core of the atp6-1 gene found in line IS1112C is absent from Tx398. Sequences further upstream are present in Tx398, but at a different genomic location. The atp6-2 genes are present in similar copy numbers in IS1112C and A3Tx398, a near-isogenic line carrying the IS1112C cytoplasm in a Tx398 background. However, transcript abundance of atp6-2 in these lines is about ten-times higher than that of atp6-1. RNA editing of atp6-1 transcripts is identical to that of atp6-2 and therefore sequence specific. A single non-silent editing site in the unique atp6-1 pre-piece sequence may indicate mitochondrial-guided RNA editing. While in Petunia the abundance and RNA editing of a transcript are correlated, we show here that RNA editing is independent of transcript abundance and is sequence specific in Sorghum.

DNA, Complementary↗
PubMed2005

Conserved spacing between the box C/D and C'/D' RNPs of the archaeal box C/D sRNP complex is required for efficient 2'-O-methylation of target RNAs.

RNA-guided nucleotide modification complexes direct the post-transcriptional nucleotide modification of both archaeal and eukaryotic RNAs. We have previously demonstrated that efficient 2'-O-methylation activity guided by an in vitro reconstituted archaeal box C/D sRNP requires juxtaposed box C/D and C'/D' RNP complexes. In these experiments, we investigate the importance of spatially positioning the box C/D and C'/D' RNPs within the sRNP complex for nucleotide modification. Initial sequence analysis of 245 archaeal box C/D sRNAs from both Eukyarchaeota and Crenarchaeota kingdoms revealed highly conserved spacing between the box C/D and C'/D' RNA motifs. Distances between boxes C to D' and C' to D (D' and D spacers, respectively) exhibit highly constrained lengths of 12 nucleotides (nt). Methanocaldococcus jannaschii sR8 sRNA, a model box C/D sRNA with D and D' spacers of 12 nt, was mutated to alter the distance between the two RNA motifs. sRNAs with longer or shorter spacer regions could still form sRNPs by associating with box C/D core proteins, L7, Nop56/58, and fibrillarin, comparable to wild-type sR8. However, these reconstituted box C/D sRNP complexes were severely deficient in methylation activity. Alteration of the D and D' spacer lengths disrupted the guided methylation activity of both the box C/D and C'/D' RNP complexes. When only one spacer region was altered, methylation activity of the corresponding RNP was lost. Collectively, these results demonstrate the importance of box C/D and C'/D' RNP positioning for preservation of critical inter-RNP interactions required for efficient box C/D sRNP-guided nucleotide methylation.

Archaeal Proteins↗