Kinetics of HTT lowering in brain of YAC 128 mice following single and repetitive intranasal dosing of siRNA packaged in chitosan-based nanoparticle.

This report describes the kinetics of Huntington's Disease (HD) gene (HTT) lowering in brains of YAC 128 mice. Lowering (or "knock-down") of HTT mRNA expression was achieved by intranasal administration of specially designed siRNA loaded into chitosan nanoparticles. Kinetic patterns of HTT lowering observed in different brain regions allowed calculation of cumulative lowering effects that result from multiple consecutive administrations. Mathematical modeling generated dosing schedules for approaching a steady knock-down effect and for prediction of magnitude and duration of HTT lowering. Kinetic modeling of HTT lowering with our algorithm will be useful in determining intranasal dosing schedules to produce chronic, therapeutically significant lowering effect of gene expression.

Gene profiling study of G3139- and Bcl-2-targeting siRNAs identifies a unique G3139 molecular signature.

G3139 is a phosphorothioate oligodeoxyribonucleotide that is targeted to the initiation codon region of the Bcl-2 mRNA, which downregulates Bcl-2 protein and mRNA expression via an antisense mechanism. In previous work, we have demonstrated that the phenotype observed in several prostate and melanoma cell lines after treatment with G3139 appears to be Bcl-2 independent. In contrast, downregulation of Bcl-2 expression by a small interfering RNA (siRNA) produced little or no phenotype change. In the present work, we performed an Agilent oligonucleotide microarray assay on mRNA isolated from PC3 prostate cancer cells that were treated with two different oligonucleotide gene-silencing reagents. G3139 and a Bcl-2-targeting siRNA both downregulate Bcl-2 expression, but via different mechanisms. A side-by-side comparative analysis showed that the expression profile generated by these molecules differs substantially. The study revealed upregulation of the expression of stress-inducible genes in PC3 cells at 1 and 3 days after a 5-h transfection with G3139 complexed with Lipofectamine 2000. In contrast, only a very diminished stress response was seen 1 and 3 days after a 24-h transfection of siRNA/Lipofectamine 2000 complexes. These results highlight the profound differences in off-target effects in cells treated with the phosphorothioate oligonucleotide G3139 and with an siRNA targeted to the same gene, and provide further evidence that the mechanism of action of G3139 is not related to Bcl-2 silencing.

Binding and disruption of phospholipid bilayers by supramolecular RNA complexes.

In an RNA world, RNAs would have regulated traffic through normally impermeable bilayer membranes. Using selection-amplification we previously found RNAs that bind stably and increase the ionic conductance of phospholipid membranes at high Mg(2+) and Ca(2+) concentrations. Now selection in reduced divalents yields RNAs that bind phosphatidylcholine liposomes under conditions closer to physiological. Such affinity for phospholipid membranes requires interactions between RNAs. In fact, we detected no functional monomeric membrane-binding RNAs. A membrane-active end-to-end heterotrimer consisting of 2 RNA 9 and 1 RNA 10 is defined by nucleotide protection, oligonucleotide competition, and mutant analysis. Oligomers of the heterotrimer bind stably, cause release of liposome-encapsulated solutes, and disrupt model black membranes. Individual RNA molecules do not show any of these activities. This novel mechanism of RNA binding to lipid membranes may not only regulate membrane permeability, but suggests that arrays of catalytic or structural RNAs on membranes are plausible. Finally, a selection met only by RNA complexes evokes new possibilities for selection-amplification itself.

Analysis of promoter recognition in vivo directed by sigma(F) of Bacillus subtilis by using random-sequence oligonucleotides.

Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase sigma factors. Of these, sigma(F) is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing sigma(F) (E-sigma(F)), and only two genes of known function are exclusively under the control of E-sigma(F), spoIIR and spoIIQ. In order to investigate the features of promoters that are recognized by E-sigma(F), we studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ. The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B. subtilis chromosome. This system made it possible to test for transcription of lacZ by E-sigma(F) in vivo. The results indicate a weak sigma(F)-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated sigma factors, as well as to sigma(A). There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated sigma factors. The looseness of the sigma(F) promoter requirement contrasts with the strict requirement for sigma(A)-directed promoters of B. subtilis. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma(F) in vivo.

The chromosomal location of the Bacillus subtilis sporulation gene spoIIR is important for its function.

Formation of the asymmetrically located septum during sporulation of Bacillus subtilis results in enclosure of the origin-proximal 30% of the chromosome in the prespore compartment. The rest of the chromosome is then translocated into the prespore from the mother cell. Transcription of spoIIR is initiated in the prespore by RNA polymerase containing sigma(F) soon after the septum is formed. The SpoIIR protein is required for the activation of the transcription program directed by sigma(E) in the mother cell. The spoIIR locus is located at 324 degrees, near the origin of replication (0/360 degrees ). We show here that movement of spoIIR to 28 degrees had little effect on sporulation. However, movement to regions not in the origin-proximal part of the chromosome substantially reduced sporulation efficiency. At 283 degrees sporulation was reduced to less than 20% of the level obtained when spoIIR was at its natural location, and movement to 190 degrees reduced sporulation to about 6% of that level. These positional effects were also seen in the transcription of a spoIIR-lacZ fusion. In contrast, movement of other spo-lacZ fusions from 28 degrees to 190 degrees had little effect on their expression. These results suggest that spoIIR is the subject of "positional regulation," in the sense that the chromosomal position of spoIIR is important for its expression and function.

Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase.

Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm. The second lacks both the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is verified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the presence of the cognate aspartate GUC sequence in the anticodon loop.

Pyrophosphate mediates the effect of certain tRNA mutations on aminoacylation of yeast tRNA(Phe).

The influence of pyrophosphate hydrolysis by inorganic pyrophosphatase on homologous aminoacylation of different yeast tRNA(Phe) mutants was studied. The addition of pyrophosphatase significantly improved the aminoacylation efficiency of tRNA(Phe) structural mutants as well as the mutant with substitution at position 20, while having no effect on the charge of wild-type tRNA(Phe). Aminoacylation of tRNA(Phe) anticodon and discriminator base (N(73)) mutants was not affected by pyrophosphatase. Activation of wild-type tRNA(Phe) transcript aminoacylation by inorganic pyrophosphatase was observed only at low Mg(2+) concentrations due to distortion of the tRNA(Phe) structure under these conditions. Our results demonstrate that pyrophosphate dissociation becomes a rate-limiting step of the reaction in yeast phenylalanyl-tRNA synthetase catalyzed aminoacylation of tRNA(Phe) variants with altered tertiary structure. A possible mechanism of pyrophosphate-mediated inhibition of tRNA mutants aminoacylation is discussed.

RNAs that bind and change the permeability of phospholipid membranes.

The RNA world hypothesis presumes that RNA will be competent for varied essential cellular functions. One such indispensable cell function is regulation of membrane permeability. Though this was not a known RNA activity, selection-amplification yielded RNAs that bound phosphatidylcholine:cholesterol liposomes. At least eight distinct, approximately 95-mer sequences bind well to the outside of the lipid bilayer, though randomized sequences had no such activity. No distinct sequence motif for lipid binding was found. However, truncation of one such RNA shows that a smaller, 44-nucleotide irregular RNA hairpin is an active membrane binding domain. Bound RNA increases the permeability of liposomes to (22)Na(+). In addition, using voltage clamp technique, four individual RNAs increase the ion permeability of the plasma membrane of cultured human cells. The existence of multiple sequences that bind membranes and provoke permeability changes suggests that these may be elementary RNA functions that could be selected in vivo.

The spoIIE locus is involved in the Spo0A-dependent switch in the location of FtsZ rings in Bacillus subtilis.

A switch in the location of FtsZ ring structures from medial to polar is one of the earliest morphological indicators of sporulation in Bacillus subtilis. This switch can be artificially caused during vegetative growth by induction of an active form, Sad67, of the transcription regulator, Spo0A (P. A. Levin and R. Losick, Genes Dev. 10:478-488, 1996). We have used immunofluorescence microscopy to show that the switch in FtsZ ring location during vegetative growth caused by Sad67 induction is blocked by a spoIIE deletion mutation. The spoIIE mutation also impaired polar FtsZ ring formation during sporulation. These results suggest that SpoIIE mediates the Spo0A-directed formation of polar FtsZ rings.

[Mechanism of discrimination of tRNA(Phe) from E. coli by yeast phenylalanine-tRNA-synthetase]. / Mekhanizm diskriminatsii tRNK(Phe) E. coli fenilalanin-tRNK-sintetazoi drozhzhei.

Some peculiarities of aminoacylation of homologous and heterologous tRNA by yeast phenylalanyl-tRNA synthetase have been studied. It was found that the enzyme is inactivated during E. coli tRNA aminoacylation due to the formation of a pyrophosphorylated form of the enzyme. Inorganic pyrophosphatase splits off the bound pyrophosphate and reactivates the enzyme; a similar effect is produced by homologous tRNA. The differences in the reaction mechanism observed with E. coli tRNA in comparison with yeast tRNA seem to be due to the sole difference in the nucleotide sequences, i. e., substitution of G-U at position 20. A putative mechanism of the enzyme inactivation during aminoacylation of heterologous tRNA is discussed.

[Pyrophosphate-dependent inactivation of tyrosyl-tRNA synthetase during aminoacylation of heterologous tRNA]. / Pirofosfatzavisimaia inaktivatsiia tirozil-tRNK sintetazy pri aminoatsilirovanii geterologichnoi tRNK.

It has been shown that heterologous aminoacylation of tRNA by tyrosyl-tRNA synthetase leads to inactivation of the enzyme. Inorganic pyrophosphatase prevents the inactivation and increases the enzyme activity and aminoacylation level in a heterologous system. A putative inactivation mechanism is discussed.

Anticodon-dependent aminoacylation of RNA minisubstrate by lysyl-tRNA synthetase.

Specific inhibition of mammalian lysyl-tRNA synthetase by polyU is shown. Inhibition of the enzyme is dependent on the length of the oligonucleotide, since oligoU molecules with a length of less than 8 residues do not inhibit the aminoacylation, whilst the effect of oligoU molecules with a length of about 30 residues is the same as that of polyU. Inhibition is a result of recognition by the enzyme of the tRNALys anticodon sequence (UUU) coded by polyU. Aminoacylation of the oligoU molecule with attached CCA sequence (G(U)20-CCA) by yeast and mammalian lysyl-tRNA synthetases is demonstrated.

Crucial role of pyrophosphate in the aminoacylation of E. coli tRNA(Phe) by yeast phenylalanyl-tRNA synthetase.

Rapid inactivation of the yeast phenylalanyl-tRNA synthetase in the course of aminoacylation of the heterologous E. coli tRNA(Phe) is observed. This inactivation occurs due to the formation of the tight complex of the enzyme with the pyrophosphate formed during the aminoacylation reaction. This complex is shown to be the normal intermediate of the reaction. Possible inactivation mechanism and correlation between structural differences of yeast and E. coli tRNAs(Phe) with the changes in the enzymatic mechanism of aminoacylation are discussed.