RNA Trans Research Articles

The Hammerhead Ribozyme Structure Brought in Line

RNA catalysis seems to be considerably more wide spread than originally thought, with the most prominent example being the ribosome, where RNA catalyses the peptidyl-transferase reaction. Among the most and longest studied catalytic RNAs are the small nucleolytic ribozymes, such as the hairpin, VS, HDV and hammerhead ribozymes. They all catalyse the site-specific cleavage of their own phosphodiester backbone in cis or that of a substrate RNA in trans through a transesterification reaction involving the 2’-OH. A novel crystal structure of the hammerhead ribozyme has just been reported, and this should help to clarify a long-standing debate on the mechanism of catalysis. First identified in the 1980s as a catalytically active element in the replication cycle of certain viroids and the satellite RNA of plant viruses, the hammerhead ribozyme is the smallest naturally occurring RNA endonuclease. The motif has also been found in transcripts from the satellite DNA of amphibians, schistosomes, cave cricket and, most recently, encoded in the genomes of other eukaryotic organisms. The hammerhead ribozyme consists of a catalytic core of 11 conserved nucleotides that are flanked by three helices (Figure 1A). In the absence of divalent metal ions, the structure is extended, but upon addition of Mg , the RNA folds in two well-defined steps into a Y-shaped structure (Figure 1B), as deduced by Lilley and coworkers in studies using comparative gel electrophoresis, FRET, NMR and calorimetry. In this active conformation, a reversible transesterification reaction is catalysed by the hammerhead ribozyme (Scheme 1). 9] During cleavage, the 2’OH of nucleotide C17 is deprotonated and attacks the scissile 3’,5’ phosphodiester bond. Of the two cleavage products one carries a 2’,3’-cyclic phosphate, the other a 5’-hydroxy terminus. In the reverse (ligation) reaction, the 5’-oxygen attacks the cyclic phosphate. For the hammerhead ribozyme, however, the ligation does not proceed as efficiently as seen for the hairpin ribozyme. Both reactions proceed through the same, trigonal-bipyramidal pentacoordinated transition state (Scheme 1), thus meeting the principle of microscopic reversibility. This transition state was deduced from the observation that the chirality of the scissile phosphate, when exchanged for a phosphorothioate, was inverted during the course of the reaction, a hallmark of the SN2 mechanism. In the transition state, the 2’-OH of C17 has to be in line with the adjacent phosphorus and the 5’-oxygen of nucleotide 1.1 (Scheme 1). This requirement and other data detailed below gave rise to presumably the longest-standing debate in the ribozyme field. The first hammerhead ribozyme crystal structures showed a maximal deviation from the ACHTUNGTRENNUNGrequired in-line orientation of the three atoms, at 908. Hammerhead cleavage, however, could be achieved by soaking all RNA crystals with divalent metal ions. While the first observation argued for a ground-state structure to be present in the crystal, the second would indicate that no major rearrangements were necessary to reach the tran[a] R. Przybilski, Dr. C. Hammann AG Molecular Interactions Department of Genetics, University of Kassel Heinrich-Plett-Strasse 40 34132 Kassel (Germany) Fax: (+49)561-804-4800 E-mail : c.hammann@uni-kassel.de Figure 1. The hammerhead ribozyme. A) Secondary structure with stems I, II and III and the 11 conserved nucleotides (bold). Cleavage takes place between nucleotides 17 and 1.1, as indicated by an arrow. Numbers are given according to the conventional scheme. In minimal versions of the ribozyme, either stem I or II is closed by loops (dashed lines). B) Y-shaped conformation of the minimal version of the ribozyme upon addition of magnesium. Naturally occurring ribozymes are endowed with tertiary stabilising structures formed between C) loops L1 and L2 or D) loop L1 and bulge B2 in stem II. The ribozyme format shown in (D) was used for crystallisation.

Evidence that PTB does not stimulate HCV IRES-driven translation

It is now well established that Hepatitis C Virus (HCV) translation is driven by an Internal Ribosome Entry Site (IRES) resulting in cap-independent translation. Such a mechanism usually occurs with the help of IRES Associated Factors (ITAFs). Moreover, an important translational feature is likely conserved from the model of classical mRNA circularisation (5'-3' cross-talk), involving the HCV RNA highly structured 3' extremity called the 3'X region. This could bind several cellular factors and modulate the translation efficacy, at least in Rabbit Reticulocyte Lysate (RRL). In particular, polypyrimidine-binding proteins have been proposed to be potential HCV ITAFs, such as Polypyrimidine Tract Binding protein (PTB). However, contradictions still exist as to the role of PTB: its ability to bind both the HCV IRES and the 3'X region leads to the hypothesis that it could positively modulate IRES-driven translation in the presence of the X structure. Results of translational and PTB-binding studies of X mutant sequences led us to discredit PTB as protagonist of 3'X region stimulation on HCV IRES-driven translation. Moreover, competition assays of X RNA in trans on IRES-driven translation demonstrate the involvement of at least two stimulating factors and led to the conclusion that this mechanism is more complex than initially thought. Although we did not identify these factors, it is no longer doubtful that there is effectively a stimulating functional interaction between the HCV IRES and the 3'X region in RRL.

HIV-1 TAR RNA Subverts RNA Interference in Transfected Cells through Sequestration of TAR RNA-binding Protein, TRBP

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.

Betanodavirus B2 Is an RNA Interference Antagonist That Facilitates Intracellular Viral RNA Accumulation

Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.

Spliced-leader RNA trans splicing in a chordate, Oikopleura dioica, with a compact genome.

trans splicing of a spliced-leader RNA (SL RNA) to the 5' ends of mRNAs has been shown to have a limited and sporadic distribution among eukaryotes. Within metazoans, only nematodes are known to process polycistronic pre-mRNAs, produced from operon units of transcription, into mature monocistronic mRNAs via an SL RNA trans-splicing mechanism. Here we demonstrate that a chordate with a highly compact genome, Oikopleura dioica, now joins Caenorhabditis elegans in coupling trans splicing with processing of polycistronic transcipts. We identified a single SL RNA which associates with Sm proteins and has a trimethyl guanosine cap structure reminiscent of spliceosomal snRNPs. The same SL RNA, estimated to be trans-spliced to at least 25% of O. dioica mRNAs, is used for the processing of both isolated or first cistrons and downstream cistrons in a polycistronic precursor. Remarkably, intercistronic regions in O. dioica are far more reduced than those in either nematodes or kinetoplastids, implying minimal cis-regulatory elements for coupling of 3'-end formation and trans splicing.

Spliceosome-mediated RNA trans-splicing in gene therapy and genomics

In human cells, the majority of the primary transcripts synthesized by RNA polymerase II contain interruptions in the coding sequences, which are known as introns. The spliceosome, a macromolecular enzyme, precisely and efficiently removes these introns in a process known as pre-messenger RNA splicing. This enzyme is also capable of recombining two RNAs in trans in a process known as spliceosome-mediated RNA trans -splicing. In this review, we highlight technologies that take advantage of this RNA trans -splicing, from gene therapy to genomics.

An Expanded CTG Trinucleotide Repeat Causes trans RNA Interference: A New Hypothesis for the Pathogenesis of Myotonic Dystrophy

Here we report a novel mechanism for the pathogenesis of myotonic dystrophy (DM). The DMPK mRNA with expanded CTG trinucleotide repeats interacts with other transcripts having expanded CAG repeats. This “trans RNA interference” occurs in vitro only when the number of CTG repeats is over 140 and the number of target CAG repeats exceeds 35. The trans RNA interference can explain all the phenomena previously reported about DM.

Ribozymes as therapeutic tools for genetic disease.

The discovery that RNA can act as a biological catalyst, as well as a genetic molecule, indicated that there was a time when biological reactions were catalysed in the absence of protein-based enzymes. It also provided the platform to develop those catalytic RNA molecules, called ribozymes, as trans -acting tools for RNA manipulation. Viral diseases or diseases due to genetic lesions could be targeted therapeutically through ribozymes, provided that the sequence of the genetic information involved in the disease is known. The hammerhead ribozyme, one of the smallest ribozymes identified, is able to induce site-specific cleavage of RNA, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Its ability to down-regulate gene expression through RNA cleavage makes the hammerhead ribozyme a candidate for genetic therapy. This could be particularly useful for dominant genetic diseases by down-regulating the expression of mutant alleles. The group I intron ribozyme, on the other hand, is capable of site-specific RNA trans -splicing. It can be engineered to replace part of an RNA with sequence attached to its 3' end. Such application may have importance in the repair of mutant mRNA molecules giving rise to genetic diseases. However, to achieve successful ribozyme-mediated RNA-directed therapy, several parameters including ribozyme stability, activity and efficient delivery must be considered. Ribozymes are promising genetic therapy agents and should, in the future, play an important role in designing strategies for the therapy of genetic diseases.

Generation of Switch Hybrid DNA Between Ig Heavy Chain-μ and Downstream Switch Regions in B Lymphocytes

AbstractIg heavy chain isotype switching in B lymphocytes is known to be preceded by transcription of a portion of the particular heavy chain gene segment that is targeted for recombination. Here, we describe an active role for these transcripts in the switch recombination process. Using an in vitro assay that exposes an artificial switch-μ (Sμ) minisubstrate to switch region transcripts in the presence of nuclear extracts from switching cells, we demonstrate that free 3′ ends of the Sμ sequence are extended onto switch region transcripts by reverse transcription. The activity was induced in splenic B lymphocytes upon activation with LPS or CD40 ligand. This in vitro process is thought to be relevant to in vivo class switching for two reasons: 1) although only one-third of the Sμ minisubstrate actually contains Sμ sequence, all crossovers between switch regions occurred in the Sμ portion; and 2) treatment of B lymphocytes with IL-4, which enriches for switching to Sγ1, increases the ratio of Sμ-Sγ1 to Sμ-Sγ3 hybrids by 16% after LPS treatment and by 37% after CD40 ligand activation, implicating this Sμ-primed reverse transcription of switch region transcripts as a novel mechanism of regulating the specificity of isotype switching. Further evidence for an active role of switch region transcripts was obtained by expressing Sα RNA in trans in the Bcl1B1 B lymphoma line. Endogenous Sμ-Sα switch circles were detected in Bcl1B1 cells expressing exogenous Sα RNA but not in mock-transfected cells.

Encapsidation of the flavivirus kunjin replicon RNA by using a complementation system providing Kunjin virus structural proteins in trans.

Kunjin virus (KUN) replicon RNA was encapsidated by a procedure involving two consecutive electroporations of BHK-21 cells, first with KUN replicon RNA C20DXrep (with prME and most of C deleted) and about 24 h later with a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressing KUN structural proteins. The presence of KUN replicon RNA in encapsidated particles was demonstrated by its amplification and expression in newly infected BHK-21 cells, detected by Northern blotting with a KUN-specific probe and by immunofluorescence analysis with anti-NS3 antibodies. No infectious particles were produced when C20DXrep RNA and recombinant SFV RNAs were electroporated simultaneously. When the second electroporation was performed with a single SFV replicon RNA expressing the KUN contiguous prME genes and the KUN C gene together but under control of two separate 26S subgenomic promoters (SFV-prME-C107), a 10-fold-higher titer of infectious particles was achieved than when two different SFV replicon RNAs expressing the KUN C gene (SFV-C107) and prME genes (SFV-prME) separately were used. No SFV replicon RNAs expressing KUN structural proteins were encapsidated in secreted particles. Infectious particles pelleted by ultracentrifugation of the culture fluid from cells sequentially transfected with C20DXrep and SFV-prME-C107 RNAs were neutralized by preincubation with monoclonal antibodies to KUN E protein. Radioimmunoprecipitation analysis with anti-E antibodies of the culture fluid of the doubly transfected cells showed the presence of C, prM/M, and E proteins in the immunoprecipitated particles. Reverse transcription-PCR analysis showed that the immunoprecipitated particles also contained KUN-specific RNA. The encapsidated replicon particles sedimented more slowly than KUN virions in a 5 to 25% sucrose density gradient and were uniformly spherical, with an approximately 35-nm diameter, compared with approximately 50 nm for KUN virions. The results of this study demonstrate for the first time packaging of flavivirus RNA in trans, and they exclude a role in packaging for virtually all of the structural region. Possible applications of the developed packaging system include the definition of the packaging signal(s) in flavivirus RNA as well as the amino acid motif(s) in the structural proteins involved in RNA encapsidation, virion assembly, and secretion. Furthermore, it could facilitate the development of a noninfectious vaccine delivery system based on encapsidation of a noncytopathic flavivirus replicon expressing heterologous genes.

A viral sequence in the 3'-untranslated region mimics a 5' cap in facilitating translation of uncapped mRNA.

For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5' cap structure [e.g. m7G(5')ppp(5')N]. We describe a translation enhancer sequence (3'TE) located in the 3'-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3'TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5' cap to this mRNA. Translation of both uncapped mRNA containing the 3'TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3'TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3'TE, like the 5' cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5'UTR was necessary for the 3'TE to function, except when the 3'TE itself was moved to the 5'UTR. Thus, the 3'TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5'UTR may serve only for the long distance 3'-5' communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3'UTR.

Myotonic dystrophy: evidence for a possible dominant-negative RNA mutation.

The trinucleotide expansion mutation causing myotonic dystrophy is in the 3' untranslated region of a protein kinase gene. The molecular mechanisms by which the expanded repeat causes the clinically variable and multisystemic disease, myotonic dystrophy, are not understood. It has been particularly difficult to rationalize the dominant inheritance with the fact that the expansion mutation lies outside of the protein-encoding gene elements, and should not be translated into protein. Here we use muscle biopsies from classical adult-onset myotonic dystrophy patients to study the accumulation of transcripts from both the normal and expanded DM kinase genes in patient muscle, and compare the results to normal and myopathic controls. We found relatively small decreases of DM kinase RNA in the total RNA pool from muscle; however, these reductions were not disease specific. Analysis of poly(A)+ RNA showed dramatic decreases of both the mutant and normal DM kinase RNAs, and these changes were disease-specific. Our findings are consistent with a novel molecular pathogenetic mechanism for myotonic dystrophy: both the normal and expanded DM kinase genes are transcribed in patient muscle, but the abnormal expansion-containing RNA has a dominant effect on RNA metabolism by preventing the accumulation of poly(A)+ RNA. The ability of the expansion mutation to alter accumulation of poly(A)+ RNA in trans suggests that myotonic dystrophy may be the first example of a dominant-negative mutation manifested at the RNA level.

Cis- and trans-acting ribozymes from a human pathogen, hepatitis delta virus

Hepatitis delta virus (HDV) contains two self-cleaving RNA sequences (ribozymes) that may naturally function as such in human cells. A pseudo-knot-containing structural motif, which is distinct from the well-characterized secondary structures of self-cleaving RNAs common to the plant pathogenic RNAs, is shared by the cis-acting HDV ribozymes. Definition of the sequences and secondary structures of the HDV ribozymes has facilitated the design of novel catalytic molecules, such as small RNA circles, capable of site-specific cleavage of RNA in trans.

Eight genes and alternative RNA processing pathways generate an unexpectedly large diversity of cytoplasmic intermediate filament proteins in the nematode Caenorhabditis elegans.

Cytoplasmic intermediate filament (IF) proteins of Caenorhabditis elegans are encoded by a dispersed multigene family comprising at least eight genes which map to three linkage groups. Exon sequences and intron patterns define three distinct subfamilies. While all eight IF genes display the long coil 1b subdomain of nuclear lamins, only six genes (a1-a4, b1 and b2) retain a lamin-like tail domain. Two genes (c1 and c2) have acquired entirely novel tail domains. The overall sequence identity of the rod domains is only 29%. The gene structures show a strong drift in number and positions of introns, none of which are common to all genes. Individual genes share only one to four intron locations with the Helix aspersa IF gene, but all eight nematode genes together account for nine of the 10 introns of the gastropod gene. All C.elegans IF genes are transcribed and all except gene c2 produce trans-spliced mRNAs. Alternatively spliced mRNAs arise from genes a1, b2 and c2 through several mechanisms acting at the transcriptional and posttranscriptional levels. These involve the alternative use of distinct promoters, polyadenylation sequences and both cis and trans RNA splice sites. The resulting sequence variations are restricted to the non-helical end domains. Minimally 12 distinct IF proteins are encoded by the various mRNAs. Different abundances in mixed-stage nematode populations suggest cell type- and/or stage-specific expression of individual mRNAs.

Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in living cells.

When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pairing with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 429 nucleotides long trans-ribozyme (2as-Rz12) which was directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occur. To study whether pre-formation of helix II in the non-paired 2as-Rz12 RNA could influence the observed cleavage rate in vitro and its inhibitory activity on HIV-1 replication, we extended the 4 base pair helix II of 2as-Rz12 to 6, 10, 21, and 22 base pairs respectively. Limited RNase cleavage reactions performed in vitro at 37 degrees C and at physiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. This modification neither affected the association rate with target RNA nor the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication in human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo.

The Conserved Terminal Guanosine of a Group I Intron can Help Prevent Reopening of the Ligated Exons

The highly conserved terminal guanosine of the Tetrahymena group I intron was mutated to an adenosine. The intron still excised itself but more slowly than the wild-type. At very low concentrations of GTP only ligated exons were produced, but at high concentrations of GTP unligated exons accumulated and the 3′ exon acquired a GTP at its 5′ end. A series of experiments suggested that GTP was primarily reopening the ligated exons, rather than directly cleaving the 3′ splice site. 5′ Truncated forms of the wild-type intron can cleave RNA in trans. Cleavage takes place downstream of sequences similar to the last few bases of the 5′ exon. The ligated exons would therefore be a potential substrate. We believe that the intron uses the terminal guanosine to compete with exogenous GTP until the ligated exons have dissociated from their binding site. Other interactions between intron sequences which are known to aid in 3′ splice-site recognition may assist in this process.

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

An in vitro system for the leader-primed transcription of coronavirus mRNAs.

We have developed an in vitro transcription system which can utilize exogenous leader RNA for mouse hepatitis virus (MHV) 'leader-primed' mRNA transcription. Cytoplasmic extracts containing viral proteins and template RNA were prepared by lysolecithin permeabilization of MHV-infected cells. Synthetic leader RNA which differed in sequence from the endogenous leader RNA was added to the extracts and demonstrated to be incorporated into MHV mRNAs. Irrespective of the size of leader RNAs added, the exogenous leader RNA was joined to the endogenous mRNA at the same site, which corresponds to a UCUAA pentanucleotide repeat region. Only leader RNAs containing the pentanucleotide sequences could be utilized for transcription. Mismatches between the intergenic site and the exogenous leader sequence within the pentanucleotide repeat region were corrected in the in vitro system. This in vitro system thus established a novel mechanism of leader-primed transcription using exogenous RNA in trans, and suggests the involvement of a specific ribonuclease activity during coronavirus mRNA synthesis.

Specific gene suppression by engineered ribozymes in monkey cells.

Short catalytic RNAs possessing specific endoribonuclease activity (ribozymes) have recently been designed that can potentially shear any chosen target RNA in trans at a specific site. Here, engineered ribozymes targeted against chloramphenicol acetyltransferase (CAT), derived from Tn9, have been cloned into a mammalian expression vector and tested in transient transfection experiments for their effects on CAT expression in monkey (COS1) cells. The ribozymes contained the catalytic domain of the satellite RNA from tobacco ringspot virus and were targeted to three sites in the CAT mRNA by flanking antisense sequences. These ribozymes, which were previously shown to accurately cleave CAT message in vitro, were cloned into a replicating plasmid vector under the control of the highly active simian virus 40 early promoter. The ribozyme gene sequence was incorporated into the 3' untranslated region of the gene for firefly luciferase as it was ineffective when expressed as a short RNA. Each ribozyme construction gave a similar level of suppression of CAT activity when the target was transcribed from the herpes virus thymidine kinase promoter. One of the three (ribozyme 2) was chosen for further study and tested after it had been modified by the addition of extra flanking bases. The reporter gene for luciferase was used to monitor ribozyme level and to function as a specificity control, and the human growth hormone gene was cotransfected as an independent reporter for specificity of the ribozyme against the intended target CAT. At high (approximately 1000-fold) molar excess this ribozyme was demonstrated to consistently and specifically suppress CAT expression (up to approximately 60%) in COS1 cells relative both to a plasmid clone with the ribozyme inserted in the reversed (inactive) orientation and to a control corresponding to the relevant 26-nucleotide antisense segment of CAT.