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T Tuschl

Publications and source records attributed to T Tuschl.

At least 19 recordsLinked to original sources
PubMed2001

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.

Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

Animals↗
PubMed2001

Identification of novel genes coding for small expressed RNAs.

In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21-nucleotide (nt) RNAs, respectively, which function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as small temporal RNAs (stRNAs). We show that many 21- and 22-nt expressed RNAs, termed microRNAs, exist in invertebrates and vertebrates and that some of these novel RNAs, similar to let-7 stRNA, are highly conserved. This suggests that sequence-specific, posttranscriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.

Animals↗
PubMed2001

A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA.

The 21-nucleotide small temporal RNA (stRNA) let-7 regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer to produce the active small-RNA component that represses gene expression.

Animals↗
PubMed2001

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

Animals↗
PubMed2001

RNA interference is mediated by 21- and 22-nucleotide RNAs.

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.

Animals↗
PubMed2001

A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation.

In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 5' exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation.

Animals↗
PubMed2001

Identification of essential genes in cultured mammalian cells using small interfering RNAs.

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both beta- and gamma-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.

Animals↗
PubMed2000

RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.

Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.

Adenosine Triphosphate↗
PubMed1999

Targeted mRNA degradation by double-stranded RNA in vitro.

Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

3' Untranslated Regions↗
PubMed1998

Selection in vitro of novel ribozymes from a partially randomized U2 and U6 snRNA library.

Combinatorial libraries related to spliceosomal U2 and U6 snRNAs were tested for catalytic reactions typical of the splicing of nuclear pre-mRNAs. Ribozymes with four different activities were selected based on covalent bond formation to a substrate RNA. The first activity was reversible self-cleavage; ribozymes self-cleaved then ligated the 5'-hydroxyl group of the substrate oligonucleotide to their 2',3'-cyclic phosphate intermediate. The second activity was 2',5'-branch formation by the attack of a substrate 2'-hydroxyl group on the 5'-terminal triphosphate of the ribozyme transcript, releasing pyrophosphate. The third ribozyme activity was similar to reversible self-cleavage but was a three-step reaction. This ribozyme self-cleaved, then cleaved the substrate in trans, and then ligated the substrate 3' cleavage product to its cyclic phosphate intermediate. This three-step pathway shares similarities with the pathway of tRNA splicing. The fourth activity was 2',3'-branch formation; to form this unusual branch, a 2'-hydroxyl of the substrate attacked an internal phosphate of the ribozyme, releasing an oligonucleotide leaving group. The isolation of branching activities by the in vitro selection protocol was unanticipated and was due to surprising properties of reverse transcriptase, which can read through 2',5'- or 2',3'-branches and efficiently perform non-templated intramolecular jumps.

Base Sequence↗
PubMed1996

Mg(2+)-dependent conformational changes in the hammerhead ribozyme.

Conformational changes of the hammerhead ribozyme were examined by fluorescence changes of 2-aminopurine riboside incorporated either in the substrate or in the ribozyme. Fluorescence changes could be observed for both the substituted substrate and ribozyme upon complex formation, indicating a different environment for the 2-aminopurine in the complex. Ribozyme-substrate constructs for ciscleavage containing 2-aminopurine at various sites were used for the determination of binding constants of Mg2+ and Ca2+. Depending upon the site of 2-aminopurine substitutions, the fluorescence intensity upon addition of Mg2+ or Ca2+ was reduced by 0-50%. The measurements were performed in high ionic strength buffers such that base pairing in the helical regions is expected to be complete. With three of the ribozymes, the dependence of the fluorescence emission as a function of Mg2+ concentration could be fitted by single binding processes, whereas for the two remaining ribozymes a second binding process needed to be included. The binding constants range from 7600 M-1 down to 12 M-1 in 75 mM Tris-HCl (pH 7.5) and indicate the presence of multiple binding sites in the ribozymes with varying degrees of affinity toward the metal ions. Mg2+ binding constants determined in the same buffer from the Mg2+ dependence of the cleavage rate are of the order of 100 M-1; thus, Mg2+ sites directly involved in catalysis are of intermediate affinity. The ribozyme containing 2-aminopurine in loop III demonstrated the highest binding constant whereas the ribozyme with a 2-aminopurine next to a 2'-deoxy-2'-aminocytosine at the cleavage site exhibited only low metal ion affinity. The data obtained for Ca2+ are very similar to those found for Mg2+. This approach provides a first set of data describing a Mg2+ binding topography to hammerhead RNA molecules and should be useful for the analysis of other RNA molecules.

2-Aminopurine↗
PubMed1995

RNA cleavage by small catalytic RNAs.

Recent studies of the hammerhead ribozyme have provided an insight into its three-dimensional structure. In addition, studies using chemical probes, functional-group modification and mutational analysis, in combination with computer modelling, have led to proposals for the structure of both the hairpin and hepatitis delta virus ribozymes. Such structural elucidations will aid understanding of the mechanism of ribozyme catalysis. The discovery that certain RNA-binding proteins can increase the catalytic efficiency of ribozymes in encouraging for their use in the inhibition of gene expression in vivo.

Animals↗
PubMed1995

Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme.

Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure.

Base Sequence↗
PubMed1994

A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements.

For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.

Base Composition↗
PubMed1994

Isoguanosine substitution of conserved adenosines in the hammerhead ribozyme.

Isoguanosine has been incorporated into a 34-mer hammerhead ribozyme by the solid-phase phosphoramidite method, using an acetamidine base protecting group. The activity of the hammerhead ribozyme when singly mutated to isoguanosine at the adenosine positions 6, 9, and 13 was 1-2-fold less than the wild-type activity. Mutations to 2-aminopurine ribonucleoside at positions 9 and 13 were 5-fold reduced in activity, but that at position 6 was approximately 30-fold reduced. These results support the view that the 6-amino functions of A6, A9, and A13 are not very important for catalysis. The 2-position of A6 tolerates a carbonyl function but not an amino group, whereas A9 and A13 tolerate both functional groups. The tolerance of a 2-amino group at A9 and A13 makes G(anti)/A(anti) Watson-Crick type base mispairing for G12/A9 and A13/G8 unlikely.

2-Aminopurine↗
PubMed1994

Oligonucleotide duplexes containing 2'-amino-2'-deoxycytidines: thermal stability and chemical reactivity.

Thermal stabilities of oligonucleotides containing 2'-amino-2'-deoxycytidines were determined and compared to those of the unmodified oligonucleotides. The presence of the 2'-aminonucleoside destabilized duplexes in a RNA as well as a DNA context at pH 7 as well as at pH 5. The pKa of the 2'-amino group was determined by 13C-NMR spectroscopy to be 6.2. The reactivity of an oligonucleotide containing a 2'-aminonucleoside was exploited for the incorporation of rhodamine by its isothiocyanate derivative.

Amines↗
PubMed1993

Activity of hammerhead ribozymes containing non-nucleotidic linkers.

Hammerhead ribozymes were synthesized in which the tetranucleotide loop II was replaced by non-nucleotidic linkers of 7, 13, 17 and 19 atoms length. Ribozymes with 17 and 19 atom linkers, in combination with a 4 base pair stem II, had catalytic efficiencies which were 2 fold increased to that of the parent ribozyme with a tetranucleotide loop. Ribozymes with these linkers, but in combination with a 2 base pair stem II, showed a 2 fold decrease in catalytic efficiency when compared to the parent ribozyme. Prolonged preincubation in the presence of MgCl2 was required for hexaethylene glycol linker-modified ribozymes to obtain maximum activity and reproducible kinetic data.

Base Sequence↗