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At least 541 records · Page 30Linked to original sources
PubMed2006

Allele-specific silencing of Alzheimer's disease genes: the amyloid precursor protein genes with Swedish or London mutations.

Alzheimer's disease (AD) is the most common cause of dementia in humans. A pathological hallmark in the brain of an AD patient is extracellular amyloid plaques formed by accumulated beta-amyloid protein (Abeta), a metabolic product of amyloid precursor protein (APP). Studies have revealed a strong genetic linkage in the early-onset familial form (<60 years old) of AD. For example, some mutant APPs are transmitted dominantly and are segregated with inheritance of early onset AD. These mutants facilitate Abeta production. The "Swedish" mutations (APP(SW)) and the "London" mutation (APP(LON)) are examples of these mutants. Selective silencing of these mutant alleles holds therapeutic promise for AD. Here we show that the expression of the mutant APPs was selectively inhibited by RNA interference. The best selectivity was obtained when the mismatches were centrally placed in the antisense strand of small interfering RNAs. Introducing an additional mismatch in the antisense strand may improve the selectivity. The addition of a G at 5' end of the antisense strand may enhance the efficacy of gene silencing by RNA interference. Our results illustrate the guiding principles for selection of targeted sequences to achieve allele-specific silencing. The sequences that are effective to silence APP(SW) and APP(LON) as identified in this study may be useful in both in vivo and in vitro studies to investigate the pathophysiological role of APP(SW) and APP(LON) in AD development.

Alleles↗
PubMedDate not supplied

Effective suppression of HIV-1 gene expression by a mammalian tRNA 3' processing endoribonuclease and external guide sequence oligozymes.

We examined the suppression of virus expression by cleaveage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.

Animals↗
PubMed2000

Effective suppression of HIV-1 gene expression by a mammalian tRNA 3' processing endoribonuclease and external guide sequence oligozymes.

We examined the suppression of virus expression by cleavage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.

Animals↗
PubMed2002

Conventional and nonconventional roles of the nucleolus.

As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to ribosome biogenesis, have been discovered within the last decade. Built around multiple copies of the genes for preribosomal RNA (rDNA), nucleolar structure is largely dependent on the process of ribosome assembly. The nucleolus is disassembled during mitosis at which time preribosomal RNA transcription and processing are suppressed; it is reassembled at the end of mitosis in part from components preserved from the previous cell cycle. Expression of preribosomal RNA (pre-rRNA) is regulated by the silencing of individual rDNA genes via alterations in chromatin structure or by controlling RNA polymerase I initiation complex formation. Preribosomal RNA processing and posttranscriptional modifications are guided by a multitude of small nucleolar RNAs. Nearly completed ribosomal subunits are exported to the cytoplasm by an established nuclear export system with the aid of specialized adapter molecules. Some preribosomal and nucleolar components are transiently localized in Cajal bodies, presumably for modification or assembly. The nonconventional functions of nucleolus include roles in viral infections, nuclear export, sequestration of regulatory molecules, modification of small RNAs, RNP assembly, and control of aging, although some of these functions are not well established. Additional progress in defining the mechanisms of each step in ribosome biogenesis as well as clarification of the precise role of the nucleolus in nonconventional activities is expected in the next decade.

Active Transport, Cell Nucleus↗
PubMed2025

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.

Animals↗
PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.

Models, Molecular↗
PubMed1997

Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs.

The majority of mitochondrial pre-mRNAs in kinetoplastid protozoa such as Trypanosoma, Leishmania, and Crithidia are substrates of a posttranscriptional processing reaction referred to as RNA editing. The process results in the insertion and, to a lesser extent, deletion of uridylates, thereby completing the informational content of the mRNAs. The specificity of the RNA editing reaction is provided by guide RNAs (gRNAs), which serve as templates for the editing apparatus. In addition, the process relies on mitochondrial proteins, presumably acting within a high-molecular-mass ribonucleoprotein complex. Although several enzymatic activities have been implicated in the editing process, no protein has been identified to date. Here we report the identification of a novel mitochondrial DEAD-box protein, which we termed mHel61p. Disruption of the mHEL61 alleles in insect-stage Trypanosoma brucei cells resulted in a reduced growth rate phenotype. On a molecular level, the null mutant showed significantly reduced amounts of edited mRNAs, whereas never-edited and nuclear mRNAs were unaffected. Reexpression of mHel61p in the knockout cell line restored the ability to efficiently synthesize edited mRNAs. The results suggest an involvement of mHel61p in the control of the abundance of edited mRNAs and thus reveal a novel function for DEAD-box proteins.

Alleles↗
PubMed2004

Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways.

In mammalian cells, both microRNAs (miRNAs) and small interfering RNAs (siRNAs) are thought to be loaded into the same RNA-induced silencing complex (RISC), where they guide mRNA degradation or translation silencing depending on the complementarity of the target. In Drosophila, Argonaute2 (AGO2) was identified as part of the RISC complex. Here we show that AGO2 is an essential component for siRNA-directed RNA interference (RNAi) response and is required for the unwinding of siRNA duplex and in consequence assembly of siRNA into RISC in Drosophila embryos. However, Drosophila embryos lacking AGO2, which are siRNA-directed RNAi-defective, are still capable of miRNA-directed target RNA cleavage. In contrast, Argonaute1 (AGO1), another Argonaute protein in fly, which is dispensable for siRNA-directed target RNA cleavage, is required for mature miRNA production that impacts on miRNA-directed RNA cleavage. The association of AGO1 with Dicer-1 and pre-miRNA also suggests that AGO1 is involved in miRNA biogenesis. Our findings show that distinct Argonaute proteins act at different steps of the small RNA silencing mechanism and suggest that there are inherent differences between siRNA-initiated RISCs and miRNA-initiated RISCs in Drosophila.

Animals↗
PubMed1993

RNA editing in mitochondria of Leishmania tarentolae and Crithidia fasciculata.

The uridine sequence of mitochondrial pre-mRNAs in trypanosomes is post-transcriptionally altered by an RNA editing process, the information for which is provided by small guide (g)RNAs. Current editing models are based on the occurrence in mitochondrial RNA of chimeric molecules in which a gRNA is covalently linked to an editing site through its 3' end. This review describes some of the work done in Leishmania tarentolae that led to the construction of these models, in comparison to data obtained in Crithidia fasciculata. In spite of the virtually complete conservation of editing patterns and genomic organization between the two species, the properties of gRNAs and chimeric molecules in C. fasciculata put the editing models in a slightly different perspective.

Animals↗
PubMed1998

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

Aldehydes↗
PubMed2004

The scrambled actin I gene in Uroleptus pisces.

The micronuclear gene encoding actin I in Uroleptus pisces occurs in two segments. Segment I contains 638 bp divided into six macronuclear destined subsegments, or MDSs, by five internal eliminated segments, or IESs. The MDSs in segment 1 are in the scrambled disorder, 1-2-4-8-6-15, with MDSs 8 and 6 inverted. Segment II contains 2452 bp divided into ten MDSs by nine IESs in the scrambled disorder, 3-5-7-10-13-12-9-14-16-11, with MDSs 12, 9, and 11 inverted. Extensive attempts by polymerase chain reaction to connect the two segments failed. We conclude that the two segments are separated by a very long IES or are in different loci. The pattern of the 16 scrambled MDSs is entirely different from the scrambled pattern observed for the actin I gene in six other stichotrichs. We conclude that the actin I gene became scrambled on two separate occasions during stichotrich evolution: once in the lineage leading to the group of six stichotrichs, which includes, among others, Sterkiella species and Stylonychia lemnae, and once in the lineage leading to Uroleptus pisces. Repeated sequence pairs (pointers) of three to 14 bases at the ends of MDSs appear to be essential for correct splicing of MDSs during macronuclear development. However, the micronuclear actin gene also contains 40 matches of eight or more bases between IESs and MDSs that do not function as pointers. To prevent these ectopic repeats from causing improper processing of the micronuclear gene appears to demand a template of DNA or RNA from the old macronucleus to guide splicing of MDSs in the orthodox order.

Actins↗
PubMed1991

Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups.

Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.

Animals↗
PubMed2025

Heritable virus-induced germline editing in tomato.

Here, we report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum). We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S (35S), S. lycopersicum ribosomal protein S5A (SlRPS5A), or S. lycopersicum YAO promoters (SlYAO). These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences. TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9. However, the progenies from the SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to the mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%. Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and from 3% to 100% at the SlDMR6, including biallelic mutations. These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE. Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.

Solanum lycopersicum↗
PubMed2006

MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism.

Since the discovery of microRNA (miRNA)-guided processing, a new type of RNA silencing, the possibility that such a mechanism could play a role in virus defense has been proposed. In this work, we have analyzed whether Plum pox virus (PPV) chimeras bearing miRNA target sequences (miR171, miR167, and miR159), which have been reported to be functional in Arabidopsis, were affected by miRNA function in three different host plants. Some of these PPV chimeras had clearly impaired infectivity compared with those carrying nonfunctional miRNA target sequences. The behaviors of PPV chimeras were similar but not identical in all the plants tested, and the deleterious effect on virus infectivity depended on the miRNA sequence cloned and on the site of insertion in the viral genome. The effect of the miRNA target sequence was drastically alleviated in transgenic plants expressing the silencing suppressor P1/HCPro. Furthermore, we show that virus chimeras readily escape RNA silencing interference through mutations within the miRNA target sequence, which mainly affected nucleotides matching the 5'-terminal region of the miRNA.

Arabidopsis↗
PubMed1995

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library.

Animals↗
PubMed2004

Pseudouridine-guide RNAs and other Cbf5p-associated RNAs in Euglena gracilis.

In eukaryotes, box H/ACA small nucleolar RNAs (snoRNAs) guide sites of pseudouridine (Psi) formation in rRNA. These snoRNAs reside in RNP complexes containing the putative Psi synthase, Cbf5p. In this study we have identified Cbf5p-associated RNAs in Euglena gracilis, an early diverging eukaryote, by immunoprecipitating Cbf5p-containing complexes from cellular extracts. We characterized one box H/ACA-like RNA which, however, does not appear to guide Psi formation in rRNA. We also identified four single Psi-guide box AGA RNAs. We determined target sites for these putative Psi-guide RNAs and confirmed that the predicted Psi modifications do, in fact, occur at these positions in Euglena rRNA. The Cbf5p-associated snoRNAs appear to be encoded by multicopy genes, some of which are clustered in the genome together with methylation-guide snoRNA genes. These modification-guide snoRNAs and snoRNA genes are the first ones to be reported in euglenid protists, the evolutionary sister group to the kinetoplastid protozoa. Unexpectedly, we also found and have partially characterized a selenocysteine tRNA homolog in the anti-Cbf5p-immunoprecipitated sample.

Animals↗
PubMed1998

A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein.

Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein-RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (i) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (ii) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites.

Binding Sites↗
PubMed1995

In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA.

Animals↗