Regulating a translational regulator: mechanisms cells use to control the activity of the fragile X mental retardation protein.

Fragile X syndrome results from the loss of a normal cellular protein, FMRP. FMRP is an RNA binding protein, and it is likely that altering the way FMRP's messenger RNA (mRNA) targets are processed results in the clinical features associated with the disease. Using complementary DNA microarray screening, a number of brain-derived mRNAs that interact directly with FMRP in vitro and associate with FMRP-containing mRNPs in vivo have been identified. These target messages encode RNA-binding proteins, transcription factors, neuronal receptors, cytoskeletal proteins, a few enzymes as well as several unknown proteins. For a subset of these mRNAs it has been shown that modulating FMRP levels in cultured cells correspondingly affects their expression. In addition, several modes by which cells modulate FMRP activity have been described; these include posttranscriptional processing and posttranslational modification. Here, the most recent results concerning the biochemical activities of FMRP and how they are affected by various modifications are reviewed. The data lead to a model signaling mechanism by which FMRP normally regulates the expression of its target mRNAs.

Selectively enriched mRNAs in rat synaptoneurosomes.

Differential display was used to identify synapse-enriched mRNAs. Of 15 mRNAs initially identified, all were found in multiple synaptoneurosome preparations; 58% were subsequently shown to be enriched in all the preparations by Northern blotting and semiquantitative RT-PCR. RNAs involved in signal transduction, vesicle trafficking, lipid modification and cell shape and remodeling were among these messages. Tip60a mRNA, recently found to associate with the fragile X mental retardation protein, was also identified. These data demonstrate the diversity of the local message pool at synapses.

Self-cleaving-ribozyme-mediated reduction of betaAPP in human rhabdomyosarcoma cells.

A self-cleaving hammerhead ribozyme targeted to codon 47 in beta-amyloid precursor protein (betaAPP) mRNA was cloned as a eucaryotic transcription cassette into the 3' UTR of enhanced green fluorescence protein (EGFP) mRNA, producing a C-terminal fusion mRNA. CMV promotor-driven vectors bearing this construct or a mutationally inactive ribozyme construct were transiently transfected into human embryonic rhabdomyosarcoma (A-204) cells and their effects studied. Ribozyme self-cleavage in vivo was demonstrated by Northern blotting and the site of self-cleavage was delineated using site-specific deoxyoligonucleotide probes and primer extension arrest. Using this ribozyme reporter we demonstrated that ribozyme expression correlated with lower betaAPP levels in the transfected cells. Control studies with the inactive ribozyme construct showed that both ribozyme cleavage and antisense mechanisms combined to produce the observed effect. Furthermore, production of truncated EGFP mRNA via ribozyme self-cleavage reduced EGFP-reporter expression compared to full-length EGFP control mRNAs, indicating that truncation affects the translatability of the reporter. This occurred because of a slight decrease in the stability of the fusion mRNA. The results of these studies suggest that self-cleaving ribozyme vectors may be an effective means of delivering and visualizing the expression of small active ribozymes in vivo.

RNAs that interact with the fragile X syndrome RNA binding protein FMRP.

The Fragile X protein FMRP is an RNA binding protein whose targets are not well known; yet, these RNAs may play an integral role in the disease's etiology. Using a biotinylated-FMRP affinity resin, we isolated RNAs from the parietal cortex of a normal adult that bound FMRP. These RNAs were amplified by differential display (DDRT-PCR) and cloned and their identities determined. Nine candidate RNAs were isolated; five RNAs, including FMR1 mRNA, encoded known proteins. Four others were novel. The specificity of binding was demonstrated for each candidate RNA. The domains required for binding a subset of the RNAs were delineated using FMRP truncation mutant proteins and it was shown that only the KH2 domain was required for binding. Binding occurred independently of homoribopolymer binding to the C-terminal arginine-glycine-rich region (RGG box), suggesting that FMRP may bind multiple RNAs simultaneously.

In vivo ribozyme targeting of betaAPP+ mRNAs.

In Alzheimer's disease (AD) and Down's syndrome (DS) patients, posttranscriptional alterations of sequences encoded by exon 9 and exon 10 of the beta-amyloid precursor protein (betaAPP) mRNA result in mutant proteins (betaAPP+) that colocalize with neurofibrillary tangles and senile plaques. These aberrant messages may contribute to the development of sporadic or late-onset Alzheimer's disease; thus, eliminating them or attenuating their expression could significantly benefit AD patients. In the present work, self-cleaving hammerhead ribozymes targeted to betaAPP exon 9 (Rz9) and betaAPP+ mutant exon 10 (Rz10) were examined for their ability to distinguish between betaAPP and betaAPP+ mRNA. In transiently transfected A-204 cells, quantitative confocal fluorescence microscopy showed that Rz9 preferentially lowered endogenous betaAPP. In contrast, in transient cotransfection experiments with betaAPP+ mRNAs containing a wild-type exon 9 and mutant exon 10 (betaAPP-9/betaAPP-10+1), or a mutant exon 9 and wild-type exon 10 (betaAPP-9+1/betaAPP-10) we found that Rz9 and Rz10 preferentially reduced betaAPP+ -mutant exon 10 mRNA in a concentration and a ribozyme-dependent manner.

Hairpin ribozyme specificity in vivo: a case of promiscuous cleavage.

We have used differential display to address the question of ribozyme specificity in vivo. Stably transfected PC12 cells bearing either a hairpin ribozyme expression plasmid targeted to betaAPP mRNA or the vector alone were analyzed using nine different primer pairs. One of the few differentially expressed genes obtained from this screen corresponded to rat ribosomal protein L19. Steady-state levels of L19 mRNA were lower in ribozyme-transfected cells compared to either vector-transfected cells or native PC12 cells, and a sequence within the L19 message was cleaved by the betaAPP hairpin ribozyme in vitro. These data imply that sequence-specific unintended cleavage of non-target mRNAs may present a formidable problem to the use of hairpin ribozyme therapeutic agents.

Facilitated reduction of beta-amyloid peptide precursor by synthetic oligonucleotides in COS-7 cells expressing a hammerhead ribozyme.

Synthetic deoxyoligonucleotides and phosphorothioate-capped oligonucleotides targeted to bases 112-128 of beta-amyloid peptide precursor (beta APP) mRNA were analyzed for their ability to reduce steady-state beta APP in COS-7 cells and in pMEP4-Rz1 cells that express a hammerhead ribozyme targeted to bases beta APP mRNA 133-148. Cells, incubated in the presence of 10 or 25 microM oligonucleotide, remained viable and morphologically identical to untreated control cells for up to 5 days. Antisense deoxyoligonucleotides beta 112C, beta 114C, and beta 116C specifically lowered beta APP in pMEP4-Rz1 cells compared to noncognate and scrambled oligonucleotide controls. The extent of the beta APP reduction did not depend on oligonucleotide length, although it did depend on the presence and proximity of the ribozyme to the oligonucleotides. beta 117N, a phosphorothioate-capped antisense oligonucleotide, also reduced beta APP levels in pMEP4-Rz1 cells; however, in this case the sense control, beta 117S, affected beta APP similarly, indicating that the observed reduction may be nonspecific. These data imply that deoxyoligonucleotides targeted immediately upstream of a ribozyme binding site can work cooperatively in vivo. Localizing the oligonucleotides and ribozyme and substrate targets to the same cellular pools further confirmed this possibility.

Use of two reverse transcriptases eliminates false-positive results in differential display.

The selection of false-positive clones in differential display PCR (DDRT-PCR) represents a formidable drawback to this otherwise powerful technique of detecting subtle differences between cell types. DDRT-PCR performed with cDNAs generated by two different reverse transcriptases from the same RNA doubles the total number of reactions; nevertheless, false-positive clones arising from small differences between RNA preparations are easily distinguished from differentially expressed transcripts.

Effects of cadmium, copper, and zinc and beta APP processing and turnover in COS-7 and PC12 cells. Relationship to Alzheimer disease pathology.

The effects of cadmium, copper, and zinc on beta APP metabolism were investigated in COS-7 and PC12 cells. Cadmium chloride (CdCl2) increased beta APP steady-state protein levels and decreased beta APP posttranslational processing. These changes were not accompanied by alterations in beta APP mRNA levels or splicing. In addition, cytosolic alpha-actin and G3PDH levels were not affected. Further, neither zinc (ZnCl2) nor copper (CuSO4) altered beta APP levels or affected its normal processing. Pulse-chase studies revealed that the rate of beta APP maturation decreased twofold in the presence of 25 microM CdCl2 compared to untreated controls. beta APP secretion from the cell also dramatically slowed. These two factors result in the accumulation of partially processed beta APP inside cells. The presence of CdCl2 also decreased the amount of an 8-kDa beta APP C-terminal fragment, indicating that the cellular compartment in which beta APP accumulates is not accessible to alpha-secretase. Studies using Brefeldin A suggest that this compartment may be the cis or medial Golgi. However, A beta production was proportionately increased. These data show that CdCl2 can modulate the beta APP cleavage to favor A beta. Finally, beta APP mis- metabolism was shown to be unrelated to the hsp70 induction elicited by CdCl2; both heat shock and CuSO4 induced hsp70 but had no effect on steady-state levels of beta APP, although heat shock did slow beta APP maturation. These data indicate that hsp70 alone cannot chaperone beta APP through an alternate processing pathway leading to A beta production.

Ribozyme and antisense RNAs inhibit coupled transcription translation by binding to rabbit polyribosomes.

The behavior of ribozyme and antisense RNAs was analyzed in a coupled rabbit reticulocyte transcription translation system. Both ribozyme and antisense RNAs were efficiently produced and bound tightly to polyribosomes at 30 degrees C, but did not produce a protein product. Antisense and ribozyme RNA binding depended upon the presence of intact ribosomes, was specific since, plasmid DNA did not associate with either ribosomes or polyribosomes, and was temperature dependent. Ribozyme-specific mRNA cleavage in the coupled system was inferred from translation inhibition studies and was confirmed by primer extension analysis. Thus, ribozyme RNA can inhibit target protein production in the coupled transcription translation system by competing out cellular mRNAs and via targeted message degradation.

Facilitator oligonucleotides increase ribozyme RNA binding to full-length RNA substrates in vitro.

Primer extension arrest (PEA) studies have demonstrated that antisense oligonucleotides (beta 112C, beta 114C), which lie upstream of a ribozyme targeted to beta-amyloid peptide precursor (beta APP) mRNA, but not sense oligonucleotides (beta 112S, beta 116S) or a scrambled oligonucleotide, beta 116 M, affect ribozyme-mediated cleavage in vitro. Substrate dissociation experiments revealed that the ribozyme binding site in this mRNA was masked; PEA kinetics showed the association of the ribozyme and substrate was enhanced by antisense oligonucleotide binding. These studies suggest that masked ribozyme cleavage sites that may occur in disease-causing mRNAs can be targeted for degradation using "facilitator" oligonucleotides.

Differential activity of trans-acting hammerhead ribozymes targeted to beta amyloid peptide precursor mRNA by altering the symmetry of helices I and III.

In order to determine whether distributing the pairing bases in helices I and III of a hammerhead ribozyme asymmetrically would enhance the cleavage of trans-acting ribozymes designed to degrade cellular mRNAs, we measured the cleavage properties of symmetric and asymmetric ribozymes targeted to the amyloid peptide precursor (beta APP) mRNA. Five ribozymes were formed from three beta APP synthetic mRNA analogs and two ribozyme RNA core sequences. Symmetric ribozymes beta 133/Rz133 and beta 125/Rz133 contained 8 bp in helix I and 7 bp in helix III. Asymmetric ribozyme beta 125/Rz125 had 13 bp in helix I and 4 bp in helix III, asymmetric ribozyme beta 123/Rz125 had 11 bp in helix I and 4 bp in helix III, while asymmetric ribozyme beta 133/Rz125 contained 8 bp in helix I and 4 bp in helix III. The ability of each ribozyme to cleave its substrate RNA was first assessed under single-turnover conditions at 37 degrees C. These studies revealed that only symmetric ribozyme, beta 133/Rz133, and asymmetric ribozyme beta 123/Rz125 effectively cleaved their substrates. Further studies using a 80 degrees C, 1-min-->37 degrees C, 1-min temperature cycling paradigm were performed to increase the cleavage efficiency of the ribozymes. Under these conditions ribozymes beta 133/Rz133, beta 125/Rz125, and beta 123/Rz125 were kinetically well behaved. Therefore, the fact that the symmetric ribozyme beta 133/Rz133 was more active than its asymmetric counterparts indicates that symmetrically distributing the pairing bases in helices I and III around this cleavage site is preferred.

Phosphorylation and accumulation of tau without any concomitant increase in tubulin levels in Chinese hamster ovary cells stably transfected with human tau441.

Eucaryotic expression vectors bearing a 1.4 kb cDNA encoding the 4 repeat isoform of human tau, tau441, in either the sense or anti-sense orientation with respect to a cytomegalovirus (CMV) promoter were constructed. The resulting constructs were used to transiently express tau in Chinese Hamster Ovary cells and to generate non-neuronal stable cell lines. Immunocytochemical studies of these cells show that tau is expressed in the sense but not the anti-sense or vector containing lines. Some of the cells expressing tau showed fine elongated processes which were stained by tau antibodies. The general tau immunostaining pattern appeared diffuse and punctuate. The expressed tau was seen both unbound and bound to microtubules. In some cells labeling with antibodies that specifically recognize hyperphosphorylation of tau was observed. The size of this population increased with increasing numbers of cell passages. However, no increase in steady-state tubulin level was observed following tau441 expression. These studies show that tau can accumulate in the cells without a concomitant increase in tubulin.

Ribozyme mediated degradation of beta-amyloid peptide precursor mRNA in COS-7 cells.

Two sets of eucaryotic expression vectors encoding trans-acting hammerhead ribozymes and trans-acting hairpin ribozymes were constructed. In one set of vectors ribozyme RNA transcription was placed under the control of a mouse mammary tumor virus long terminal repeat (MMTV-LTR). In the other set ribozyme expression was controlled by a metallothionein IIA (Mt-IIA) promoter. Each ribozyme was directed to the first target sequence in the Alzheimer amyloid peptide precursor mRNA (beta APP mRNA), 5' decreases GUC decreases 3'. Ribozyme RNA transcribed from these vectors, which should cleave all six alternatively spliced forms of beta APP mRNA as well as beta APP pre-mRNA, was shown to cleave a beta APP RNA substrate analog in vitro. Stably transfected COS-7 cell lines bearing both vector types were prepared. Steady-state levels of beta APP mRNA were reduced 25-30% in cells containing either active or mutant hammerhead ribozyme vectors driven by the MMTV-LTR promoter grown in the presence of glucocorticoids. In cell lines bearing Mt-IIA driven ribozymes steady-state levels of beta APP mRNA were reduced 67-80% in both hammerhead and hairpin ribozyme containing cell lines following promoter induction by glucocorticoids. These levels correlate with the appearance of low levels of induced ribozyme RNA. In contrast, steady-state alpha-actin mRNA and G3PDH mRNA levels in these cells remained constant. Western blotting of cell extracts revealed that all forms of beta APP were correspondingly reduced. Neither the RNA nor protein decreases observed in ribozyme transfected cell lines were observed in stably transfected control cells bearing the vector alone. These results suggest that ribozyme-mediated degradation of beta APP mRNA in COS-7 cells does not depend on ribozyme cleavage.

Using RNAFOLD to predict the activity of small catalytic RNAs.

A computer-assisted method for screening the relative activity of small catalytic RNAs is described. A series of four cis-acting variant sequence files are constructed based upon the RNA sequence of a trans-acting catalytic RNA under consideration. These files are individually folded into their lowest energy structure using a standard RNA folding algorithm. The compiled structures are assessed for their ability to form recognizable catalytic RNA secondary structure motifs. We demonstrate here that RNA sequences that do not readily fold into a cis-acting hammerhead ribozyme motif have trans-acting counterparts with reduced in vitro activity. This procedure should be useful in selecting the best candidate sequences in an mRNA to target for catalytic RNA cleavage.

Cleavage of full-length beta APP mRNA by hammerhead ribozymes.

The sequences surrounding the first 5'GUC3' in the mRNA encoding the Alzheimer amyloid peptide precursor (beta APP) were used to construct a pair of transacting hammerhead ribozymes. Each ribozyme contained the conserved core bases of the hammerhead motif found in the positive strand of satellite RNA of tobacco ringspot virus [(+)sTRSV] and two stems, 7 and 8 bases long, complementary to the target, beta APP mRNA. However, one of the ribozyme cleaving strands was lengthened at its 3' end to include the early splicing and polyadenylation signal sequences of SV40 viral RNA. This RNA, therefore, more closely mimics transcripts produced by RNA polymerase II from eucaryotic expression vectors in vivo. RNA, prepared by run-off transcription of cDNA oligonucleotide or plasmid constructs containing a T7 RNA polymerase promoter was used to characterize several properties of the cleavage reaction. In the presence of both ribozyme cleaving strands magnesium-ion dependent cleavage of a model 26 base beta APP substrate RNA or full-length beta APP-751 mRNA was observed at the hammerhead consensus cleavage site. Neither ribozyme was active against non-message homologs of beta APP mRNA, nor was cleavage detected when point mutations were made in the conserved core sequences. However, the kcat/Km at 37 degrees C in 10 mM Mg+2 of the longer ribozyme was reduced twenty-fold when model and full-length substrates were compared. The use of short deoxyoligonucleotides (13-17 mers) that bind upstream of the ribozyme was found to enhance the rate of cleavage of the full-length but not beta APP model substrate RNAs. The rate of enhancement depended on both the length of the deoxyoligonucleotide used as well as its site of binding with respect to the ribozyme. These data demonstrate the utility of ribozymes to cleave target RNAs in a catalytic, site-specific fashion in vitro. Direct comparison of the efficiency of different ribozyme constructs and different modulating activities provide an experimental strategy for designing more effective ribozymes for therapeutic purposes.

A system for studying the effect(s) of familial Alzheimer disease mutations on the processing of the beta-amyloid peptide precursor.

Three different point mutations have been observed in some familial Alzheimer's disease pedigrees at a unique valine, Val717, near the carboxyl end of the beta Amyloid Peptide Precursor (beta APP). The effects of these mutations on the processing and cellular functions of beta APP can best be determined in the absence of the normal form(s) of the protein. We have used targeted mRNA degradation by a trans-acting hammerhead ribozyme to cleave and inactivate beta APP expression in vitro. The consensus ribozyme cleavage site, 5'GUC decreases X3, matches the Val717 nucleotide sequence in beta APP mRNA. Introduction of FAD point mutations which change Val717 decrease the rate of ribozyme cleavage by more than three orders of magnitude. Thus, ribozyme targeting of this site should allow the study of protein processing in vivo. Furthermore, a ribozyme targeted to mutant beta APP mRNA (Val717-->Ile) cleaved the mutant sequence 300-fold faster than the normal sequence. This suggests that ribozymes might lower mutant beta APP mRNA levels in FAD cells.

Hammerhead ribozyme cleavage of hamster prion pre-mRNA in complex cell-free model systems.

The cleavage properties of a trans-acting hammerhead ribozyme targeted 51 bases upstream of the putative splicing branch point in the hamster prion pre-mRNA intron were investigated in cell-free model systems in vitro. The specificity of cleavage was demonstrated by the inability of this ribozyme to cleave a non-homologous synthetic message encoding part of the beta amyloid peptide precursor, beta APP, and by the inability of the prion pre-mRNA to be cleaved by a ribozyme targeted to beta amyloid peptide precursor mRNA. Also, the addition of total RNA isolated from rat brain had only a minimal effect on the cleavage of the prion substrate pre-mRNA by the ribozyme. Finally neither the presence of 100 ng of nuclear or cytoplasmic proteins were found to affect the rate of cleavage in vitro.

Association-dissociation of mammalian brain glutamine synthetase: effects of metal ions and other ligands.

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn4E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982) Biochemistry 24, 6389-6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 micrograms/ml (20 nM octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn4EMn4 could be activated further by Mn(II) to form a species Mn4EMn4Mn8 that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn4EMn4, associated to octamers more extensively than Mn4E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and L-glutamine, or ATP and L-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a Kd value of ca. 2.5 X 10(-6) M, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 +/- 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 M urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (+/- 20) for tetramers in solution. Light-scattering experiments showed that, with 1.7-2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn4EMn4Mn8)n, where n greater than or equal to 5. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer.(ABSTRACT TRUNCATED AT 400 WORDS)