Sequence elements outside the catalytic core of natural hairpin ribozymes modulate the reactions differentially

2011 ◽  
Vol 392 (7) ◽  
Author(s):  
Preeti Bajaj ◽  
Gerhard Steger ◽  
Christian Hammann
Keyword(s):  
Mutational Analysis ◽  
Hammerhead Ribozyme ◽  
The Other ◽  
Hairpin Ribozyme ◽  
Catalytic Core ◽  
Arabis Mosaic Virus ◽  
Sequence Elements ◽  
Satellite Rnas ◽  
Hairpin Ribozymes

AbstractHairpin ribozymes occur naturally only in the satellite RNAs of tobacco ringspot virus (TRsV), chicory yellow mottle virus (CYMoV) and arabis mosaic virus (ArMV). The catalytic centre of the predominantly studied sTRsV hairpin ribozyme, and of sArMV is organised around a four-way helical junction. We show here that sCYMoV features a five-way helical junction instead. Mutational analysis indicates that the fifth stem does not influence kinetic parameters of the sCYMoV hairpin ribozymein vitroreactions, and therefore seems an appendix to that junction in the other ribozymes. We report further that all three ribozymes feature a three-way helical junction outside the catalytic core in stem A, with Watson-Crick complementarity to loop nucleotides in stem B. Kinetic analyses of cleavage and ligation reactions of several variants of the sTRsV and sCYMoV hairpin ribozymesin vitroshow that the presence of this junction interferes with their reactions, particularly the ligation. We provide evidence that this is not due to a presumed interaction of the afore-mentioned elements in stems A and B. The evolutionary survival of thiscis-inhibiting element seems rather to be caused by the coincidence of its position with that of the hammerhead ribozyme in the other RNA polarity.

Catalytic Properties of Hairpin Ribozymes Derived from Chicory Yellow Mottle Virus and Arabis Mosaic Virus Satellite RNAs

Biochemistry ◽  
1995 ◽  
Vol 34 (48) ◽  
pp. 15785-15791 ◽  
Author(s):  
Mary Beth DeYoung ◽  
Andrew M. Siwkowski ◽  
Ying Lian ◽  
Arnold Hampel
Keyword(s):  
Mosaic Virus ◽  
Catalytic Properties ◽  
Mottle Virus ◽  
Arabis Mosaic Virus ◽  
Yellow Mottle ◽  
Satellite Rnas ◽  
Hairpin Ribozymes

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity

2003 ◽  
Vol 10 (9) ◽  
pp. 708-712 ◽  
Author(s):  
Anastasia Khvorova ◽  
Aurélie Lescoute ◽  
Eric Westhof ◽  
Sumedha D Jayasena
Keyword(s):  
Hammerhead Ribozyme ◽  
Catalytic Core ◽  
Intracellular Activity ◽  
Sequence Elements

In Vitro Activity of the Hairpin Ribozyme Derived from the Negative Strand of Arabis Mosaic Virus Satellite RNA

1997 ◽  
Vol 122 (2) ◽  
pp. 352-357 ◽  
Author(s):  
S. Hisamatsu ◽  
Y. Morikawa ◽  
R. Tomita ◽  
T. Tanaka ◽  
S. Sonoki ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Hairpin Ribozyme ◽  
Satellite Rna ◽  
Arabis Mosaic Virus ◽  
Negative Strand

scholarly journals Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (6) ◽  
pp. 2523-2535 ◽  
Author(s):  
J H Hegemann ◽  
J H Shero ◽  
G Cottarel ◽  
P Philippsen ◽  
P Hieter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Vector System ◽  
Chromosome Loss ◽  
Wild Type ◽  
Sequence Elements ◽  
Chromosome Fragments

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

Marked inhibition of glioblastoma target cell tumorigenicity in vitro by retrovirus-mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor messenger RNA

2000 ◽  
Vol 92 (2) ◽  
pp. 297-305 ◽  
Author(s):  
Marc-Eric Halatsch ◽  
Ursula Schmidt ◽  
Ingolf C. Bötefür ◽  
James F. Holland ◽  
Takao Ohnuma
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Target Cell ◽  
Deletion Mutant ◽  
Hairpin Ribozyme ◽  
Content Type ◽  
Epidermal Growth ◽  
Hairpin Ribozymes ◽  
Fine Print

Object. The goal of this study was to evaluate the activity of certain hairpin ribozymes against deletion-mutant epidermal growth factor receptor (ΔEGFR) messenger (m)RNA in glioblastomas multiforme (GBMs). A distinct 801-bp deletion mutation associated with amplification of the EGFR gene is present in a large subgroup of primary GBMs and confers enhanced tumorigenicity in vivo. As a result of the deletion mutation, the fusion junction of the gene is created directly upstream of a GTA triplet, which is subsequently transcribed into a ribozyme target codon (GUA).Methods. In attempts to intercept ΔEGFR gene expression at the mRNA level, the authors designed three different hairpin ribozymes derived from the negative strands of satellite RNAs in tobacco ringspot virus, chicory yellow mottle virus (sCYMV1), and arabis mosaic virus against this target and evaluated their efficiency and specificity in a cell-free system. The sCYMV1, identified as the most active anti-ΔEGFR hairpin ribozyme motif, was cloned into the retroviral plasmid N2A+tRNAimet. High-titer recombinant retrovirus-containing supernatants (> 105 colony-forming units/ml) derived from an amphotropic GP+envAM 12 packaging cell line transfected with the N2A+tRNAimet-anti-ΔEGFR-sCYMV1 construct were used to introduce the sCYMV1 hairpin ribozyme into U-87MG.ΔEGFR glioblastoma cells, which overexpress exogenous ΔEGFR. Using a virus/target cell ratio of 40:1 in the absence of drug selection, the ribozyme transfer resulted in a greater than 90% reduction of ΔEGFR mRNA levels, a 69% inhibition of ΔEGFR-mediated proliferation advantage, and a greater than 95% decrease of colony formation in soft agar under relative serum starvation conditions in vitro; transfer of a control mutant ribozyme that was rendered incapable of cleaving its target yielded none of these effects.Conclusions. These findings indicate that the anti-ΔEGFR-sCYMV1 hairpin ribozyme is capable of specifically inhibiting the expression of ΔEGFR and reversing the ΔEGFR-associated malignant phenotype of GBM cells. This strategy may constitute a promising gene therapy approach for a molecularly defined subgroup of GBMs.

scholarly journals Erratum: Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity

2003 ◽  
Vol 10 (10) ◽  
pp. 872-872 ◽  
Author(s):  
Anastasia Khvorova ◽  
Aurélie Lescoute ◽  
Eric Westhof ◽  
Sumedha D Jayasena
Keyword(s):  
Hammerhead Ribozyme ◽  
Catalytic Core ◽  
Intracellular Activity ◽  
Sequence Elements
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