mRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes

Lee, Nan Sook; Bertrand, Édouard; Rossi, John J.

doi:10.1017/s1355838299990246

Cited by 36 publications

(18 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The proximity-dependent trans-splicing is analogous to previous studies where we and others have demonstrated colocalization of catalytic RNAs with their substrate RNAs inside cells enhances the activity of ribozymes (Sullenger and Cech 1993;Lee et al 1999Lee et al , 2001). For example, the anti-viral activity of ribozymes is enhanced by appending localization signals to the ribozymes to sort them to the same location as their viral target RNAs (Sullenger and Cech 1993;Lee et al 1999Lee et al , 2001. By contrast, in this study, we observed another subset of premRNAs can serve as substrates for trans-splicing regardless of the expression site of the PTM and pre-mRNA transcripts (Tables 4, 5).…”

Section: Discussionsupporting

confidence: 79%

Proximity-dependent and proximity-independent trans-splicing in mammalian cells

Viles

Sullenger²

2008

RNA

View full text Add to dashboard Cite

Most human pre-mRNAs are cis-spliced, removing introns and joining flanking exons of the same RNA molecule. However, splicing of exons present on separate pre-mRNA molecules can also occur. This trans-splicing reaction can be exploited by pretrans-splicing molecules (PTMs), which are incapable of cis-splicing. PTM-mediated trans-splicing has been utilized to repair mutant RNAs as a novel approach to gene therapy. Herein we explore how the site of PTM expression influences trans-splicing activity. We stably inserted a PTM expression cassette into the genome of HEK293 cells, generating clonal lines with single, unique insertion sites. We analyzed trans-splicing to the gene where the PTM was integrated, as well as genes neighboring these loci. We observed some pre-mRNAs only serve as substrates for trans-splicing when they are expressed in immediate proximity to the PTM expression site. The need for PTMs to be in close proximity with pre-mRNAs to trans-splice with them is consistent with the observation that pre-mRNA cis-splicing occurs cotranscriptionally. Interestingly, we identified several cellular premRNAs in one localized area that serve as trans-splicing substrates irrespective of the PTM expression site. Thus, we find multiple cellular pre-mRNAs require PTM expression in close proximity to trans-splice while others do not.

show abstract

Section: Discussionsupporting

confidence: 79%

Proximity-dependent and proximity-independent trans-splicing in mammalian cells

Viles

Sullenger²

2008

RNA

View full text Add to dashboard Cite

show abstract

“…The expression of identical localization signals within the 3′ UTR of each transcript has been demonstrated to colocalize the RNAs and improve reaction efficiency compared with nonlocalized RNAs (23). This strategy has yet to be investigated for the trans-splicing ribozyme.…”

Section: Discussionmentioning

confidence: 99%

Functional repair of a mutant chloride channel using a trans-splicing ribozyme

Rogers¹,

Vanoye²,

Sullenger³

et al. 2002

J. Clin. Invest.

View full text Add to dashboard Cite

IntroductionAdvances in molecular genetics have fostered the development of new therapeutic approaches, such as genebased therapy, for the treatment of inherited disorders. The most widely used strategy involves the introduction of wild-type sequence into cells with a defective gene. This strategy is best suited for the treatment of inherited diseases in which there is complete absence of functional alleles, such as in recessive disorders. However, it is not clear that this approach will succeed in all disorders, especially those with a dominant-negative mechanism of disease pathogenesis.Strategies using repair of mRNA are alternative approaches that will conceptually work in both dominant and recessive disorders (1). Replacing the defective portion of an mRNA molecule reduces the fraction of mutant transcripts and increases the absolute level of the normal (or repaired) transcript. The advantages of this strategy are that the target mRNA remains under the control of its endogenous promoter, thereby assuring proper regulation of expression, and tissue specificity is achieved by targeting mRNA only in cells expressing the mutant gene. RNA repair has been accomplished by two similar methods: spliceosome-mediated (2) and ribozyme-mediated trans-splicing (3-6).The Tetrahymena group I intron ribozyme has been modified to facilitate trans-splicing of its 3′ exon onto separate RNA molecules in a sequence-specific manner (7,8). This trans-splicing reaction is initiated by complementary base pairing of the target RNA to the ribozyme internal guide sequence (IGS). The ribozyme then catalyzes the cleavage of the target RNA immediately downstream of the IGS binding site and the subsequent ligation of its 3′ exon to the remaining target RNA, generating a complete repaired RNA transcript. Ribozyme-mediated transsplicing has been used to repair defective β-globin (9), myotonic dystrophy protein kinase (10), and p53 transcripts (11) in cultured mammalian cells. To date, the longest trans-spliced 3′ exon has been 1.1 kb (11), but the ability of the Tetrahymena ribozyme to mediate trans-splicing of larger exons has not been determined. Furthermore, the efficiency of the trans-splicing reaction has not been accurately measured in a cell-based system. Importantly, repaired transcripts must be translated into functional protein, but only one study has demonstrated restored protein function determined indirectly in lysates from a population of cells (11).To further investigate the feasibility of RNA repair, we designed and tested the ability of a modified Tetrahymena ribozyme to mediate trans-splicing repair of a mutant canine skeletal muscle chloride channel (cClC-1) mRNA transcript that causes the inherited disorder myotonia congenita. We demonstrated that this ribozyme can mediate trans-splicing of a large (4 kb) 3′ exon in cultured cells. Furthermore, we measured trans-splicing reaction

show abstract

“…We have previously shown that a M1GS ribozyme cleaved the mRNA sequence encoding the thymidine kinase (TK) of herpes simplex virus 1 (HSV-1) (19,20). When the ribozyme was expressed in mammalian cells infected with HSV-1, both the viral TK mRNA and protein levels were reduced by 75% (19).While little is known about the rate-limiting step of M1GS RNA cleavage reaction in cultured cells, studies on hammerhead and hairpin ribozymes suggest that binding of the ribozyme to its target RNA appears to be rate-limiting in vivo (4,9,21,22). Extensive studies have been carried out to develop strategies to express these ribozymes that colocalize with the substrates within a particular cellular compartment, and to design ribozymes to target the mRNA regions that are accessible to binding (4,9,(21)(22)(23)(24).…”

mentioning

confidence: 99%

“…While little is known about the rate-limiting step of M1GS RNA cleavage reaction in cultured cells, studies on hammerhead and hairpin ribozymes suggest that binding of the ribozyme to its target RNA appears to be rate-limiting in vivo (4,9,21,22). Extensive studies have been carried out to develop strategies to express these ribozymes that colocalize with the substrates within a particular cellular compartment, and to design ribozymes to target the mRNA regions that are accessible to binding (4,9,(21)(22)(23)(24).…”

mentioning

confidence: 99%

“…Extensive studies have been carried out to develop strategies to express these ribozymes that colocalize with the substrates within a particular cellular compartment, and to design ribozymes to target the mRNA regions that are accessible to binding (4,9,(21)(22)(23)(24). These studies have led to significant improvements of the efficacies of hammerhead and hairpin ribozymes in cellular environments.…”

mentioning

confidence: 99%

See 1 more Smart Citation

RNase P Ribozymes Selected in Vitro to Cleave a Viral mRNA Effectively Inhibit Its Expression in Cell Culture

Kilani¹,

Trang

et al. 2000

Journal of Biological Chemistry

151

View full text Add to dashboard Cite

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G 224 G 225 3 AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k cat /K m ) than that derived from the wild type ribozyme. Our results suggest that the mutated A 224 A 225 are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.RNA enzymes are being developed as promising gene-targeting reagents to specifically cleave RNA sequences of choice (1-3). For example, both hammerhead and hairpin ribozymes have been shown to cleave viral mRNA sequences and inhibit viral replication in cells infected with human viruses, while a ribozyme derived from a group I intron has been used to repair mutant mRNAs in cells (4 -9). Thus, ribozymes can be used as a tool in both basic and clinical research, such as in studies of tumorigenesis and antiviral gene therapy.RNase P is a ribonucleoprotein complex responsible for the 5Ј maturation of tRNAs (10, 11). It catalyzes a hydrolysis reaction to remove a 5Ј leader sequence from tRNA precursors (ptRNA) 1 and several other small RNAs . In Escherichia coli, RNase P consists of a catalytic RNA subunit (M1 RNA) and a protein subunit (C5 protein) (10, 11). In the presence of a high concentration of salt, such as 100 mM Mg 2ϩ , M1 RNA acts as a catalyst and cleaves ptRNAs in vitro in the absence of C5 protein (12). Extensive studies with both phylogenetic and biochemical analyses have established models for the secondary and threedimensional structures of RNase P catalytic RNAs (13-16). These models provide a framework to identify the putative active site and substrate binding site, and to study the mechanism of RNase P catalytic RNAs.Studies on substrate recognition by RNase P have revealed that a small model substrate can be cleaved efficiently by M1 ribozyme (Fig. 1A). This model substrate contains a structure equivalent to the acceptor stem, the T-stem, the 3Ј CCA sequence, and the 5Ј leader sequence of a ptRNA molecule. Accordingly, M1 catalytic RNA can cleave a mRNA sequence if the mRNA substrate forms a hybrid complex with its complementary sequence (external guide sequence) (Fig. 1A) (17). Moreover, a sequen...

show abstract

mRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes

Abstract: ABSTRACT

Cited by 36 publications

References 41 publications

Proximity-dependent and proximity-independent trans-splicing in mammalian cells

Proximity-dependent and proximity-independent trans-splicing in mammalian cells

Functional repair of a mutant chloride channel using a trans-splicing ribozyme

RNase P Ribozymes Selected in Vitro to Cleave a Viral mRNA Effectively Inhibit Its Expression in Cell Culture

Contact Info

Product

Resources

About