The c4 repressor of bacteriophage P1 is a processed 77 base antisense RNA.

The c4 repressors of the temperate bacteriophages P1 and P7 inhibit antirepressor synthesis and are essential for establishment and maintenance of lysogeny. Using in vivo complementation tests we have previously shown that c4 is an antisense RNA acting on a target, ant mRNA, which is transcribed from the same promoter. Here we identify the c4 repressor molecule of P1 as a 77 +/- 1 base RNA by mapping its termini and show that the c4 RNA in P7 lysogens has the same or a similar size. P1 c4 RNA is encoded in a region shown to be sufficient for c4 complementation. It covers exactly the 74 bases previously suggested to fold into a stem-loop secondary structure essential for c4 function. Furthermore, we demonstrate that the 5' end of c4 RNA is generated by processing. Thus, c4 is the first example of an antisense RNA to be processed. A possible mechanism of processing is discussed.     

Second-site suppressors of the bacteriophage P1 virs mutant reveal the interdependence of the c4, icd, and ant genes in the P1 immI operon.

The immI operon of phage P1 contains the genes c4, icd, and ant, which are transcribed in that order from the same constitutive promoter, P51b. The gene c4 encodes an antisense RNA which inhibits the synthesis of an antirepressor by acting on a target ant mRNA. Interaction depends on the complementarity of two pairs of short sequences encompassing virs+ and the ribosome-binding site involved in ant expression. Accordingly, in a P1 virs mutant phage, antirepressor is synthesized constitutively. We have isolated lysogen-proficient, second-site suppressors of P1 virs in order to evaluate the interdependence of the immI-specific genes. From a total of 17 suppressors analyzed, 15 were found to be located in the icd gene. They were identified as frameshift mutations, containing base insertions or deletions in tandem repeats of a single base pair. One suppressor was identified as a P51b promoter-down mutation; the second site of another suppressor was found to be located in the c4 gene. Furthermore, it was shown that virs cannot be suppressed by ant (icd+) suppressors. The results confirm the model that the immI operon is transcribed as a unit, that the icd and ant genes are translationally coupled, and that the constitutive synthesis of Icd protein alone is lethal to the bacterial cell. The existence of a c4 suppressor of virs, whose effect is not yet known, points to a still more complex regulation of antirepressor synthesis than was anticipated from the model.     

Antisense RNA control of gene expression in bacteriophage P22. II. Kinetic mechanism and cation specificity of the pairing reaction.

The bacteriophage P22 sar RNA-ant mRNA pairing reaction was characterized kinetically. The pairing reaction proceeds by a three-step pathway. First, reversible base pairs form between complementary hairpin loops in sar RNA and ant RNA (Kd = 270 nM). Next, stable duplex formation initiates between single-stranded nucleotides in sar RNA and ant RNA; the ant RNA nucleotides are at the bottom of a hairpin stem that is partially accessible. Concomitant unwinding of one sar RNA hairpin and the complementary ant RNA hairpin then occurs, to form a partially paired intermediate (k2 = 12 min(-1). Finally, a complete duplex forms after unwinding of the other sar RNA hairpin and the complementary ant RNA hairpin (k3 = 7 min(-1). Experiments with sar RNA sequence and length variants demonstrate that the precise structures of both sar RNA hairpins affect the kinetic parameters. The pairing reaction is Mg2+-dependent, and shows high specificity for the required cation. Maximal pairing rates are achieved when more than one Mg2+ ion is bound. The cation-dependence and specificity indicate a requirement for Mg2+-dependent tertiary structure.     

Inhibition of c-erbA mRNA splicing by a naturally occurring antisense RNA.

The rat erbA alpha locus encodes two overlapping mRNAs, alpha 1 and alpha 2, which are identical except for their most 3' exons. alpha 1 mRNA encodes a thyroid hormone receptor, while alpha 2 encodes an altered ligand binding domain of unknown function. Previous studies have shown that the ratio of alpha 1 to alpha 2 is highest in cells expressing a high level of a third RNA, Rev-ErbA alpha mRNA, which is transcribed in the opposite direction and is complementary to alpha 2 but not alpha 1 mRNA. It was hypothesized that base pairing with Rev-ErbA alpha blocks splicing of alpha 2 mRNA, thereby favoring formation of the non-overlapping alpha 1. To test this model, a system was developed in which alpha 2 pre-mRNAs were accurately spliced in vitro. Splicing was inhibited by the addition of a 5-fold excess of antisense RNAs containing the 3' end of Rev-ErbA alpha mRNA. Both an antisense RNA extending across the 3' splice site and a shorter RNA complementary only to exon sequences efficiently blocked splicing. However, splicing was only inhibited by complementary RNAs. These observations are consistent with a mechanism in which base pairing with a complementary RNA regulates alternative processing of alpha 1 and alpha 2 mRNAs.     

Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences

Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.     

Antisense RNA control of gene expression in bacteriophage P22. I. Structures of sar RNA and its target, ant mRNA.

Sar RNA is an antisense RNA that is partly responsible for the negative regulation of antirepressor synthesis during development of bacteriophage P22 (Liao SM et al., 1987, Genes & Dev 1:197-203; Wu Th, Liao SM, McClure WR, Susskind MM, Genes & Dev 1:204-212). The structures of sar RNA and its target, ant mRNA, were probed using limited RNase digestion as a function of Mg2+ concentration. Sar RNA forms two hairpins that are present at all Mg2+ concentrations (Mg2+-independent hairpins). One of the hairpins contains three tandem U x U base pairs. Ant RNA forms three Mg2+-independent hairpins and one Mg2+-dependent hairpin. In addition, many nucleotides in sar RNA and ant RNA appear to be involved in tertiary interactions. The effects of RNA structure on the pairing reaction are considered in the accompanying paper (Schaefer KL, McClure WR, 1997, RNA 3:157-174).     

Fast and accurate determination of sites along the FUT2 in vitro transcript that are accessible to antisense oligonucleotides by application of secondary structure predictions and RNase H in combination with MALDI-TOF mass spectrometry

Alteration of gene expression by use of antisense oligonucleotides has considerable potential for therapeutic purposes and scientific studies. Although applied for almost 25 years, this technique is still associated with difficulties in finding antisense-effective regions along the target mRNA. This is mainly due to strong secondary structures preventing binding of antisense oligonucleotides and RNase H, playing a major role in antisense-mediated degradation of the mRNA. These difficulties make empirical testing of a large number of sequences complementary to various sites in the target mRNA a very lengthy and troublesome procedure. To overcome this problem, more recent strategies to find efficient antisense sites are based on secondary structure prediction and RNase H-dependent mechanisms. We were the first who directly combined these two strategies; antisense oligonucleotides complementary to predicted unpaired target mRNA regions were designed and hybridized to the corresponding RNAs. Incubation with RNase H led to cleavage of the RNA at the respective hybridization sites. Analysis of the RNA fragments by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which has not been used in this context before, allowed exact determination of the cleavage site. Thus the technique described here is very promising when searching for effective antisense sites.     

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.     

Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect

Introduction of a constitutive antisense full-length chalcone synthase (CHS) cDNA gene in petunia can result in an inhibition of flower pigmentation. We have evaluated some of the factors which may be important for the effectiveness of an antisense CHS gene.Antisense CHS genes encoding half-length or quarter-length RNA complementary to the 3 half of CHS mRNA are able to affect flower pigmentation, while a gene encoding RNA complementary to the 5 half of CHS mRNA did not show phenotypic effects in transgenic petunia plants. We demonstrate that the RNA encoded by the latter gene has a much lower average steady-state level in leaf tissue than the RNAs encoded by the other antisense gene constructs. We have compared the CaMV 35S and endogenous CHS promoter strengths and intrinsic stabilities of sense and antisense CHS RNAs. From the data we conclude that the constitutive antisense CHS genes are not likely to provide an excess of antisense RNA compared to the CHS mRNA derived from the endogenous genes.Effective inhibition of flower pigmentation is also observed when the antisense CHS gene is under control of the homologous CHS promoter. The results indicate that the mechanism of antisense inhibition cannot solely operate via RNA duplex formation between sense and antisense RNA.     

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to exons.

The objective of the experiments described in this paper was to determine the feasibility of inhibition of pre-mRNA splicing by antisense RNA in vitro. Three different types of antisense RNA were utilized: antisense RNA complementary to the spliced RNA molecule; antisense RNA complementary to the unprocessed mRNA precursor molecule; and antisense RNA complementary to the 5' and 3' splice junctions. Whereas antisense RNA complementary to mRNA had little effect on splicing, antisense RNAs complementary to mRNA precursor or to splice junctions strongly inhibited splicing of pre-mRNA molecule. The results obtained indicate that the inhibitory effect is most likely due to hybrid formation between pre-mRNA and antisense RNA molecules and that antisense RNA complementary to the exon portion but not to the intron portion of splice junction exhibit an inhibitory effect. This inhibition can be overcome by bringing together 5' and 3' splice junctions via hybrid formation with antisense RNA complementary to the spliced RNA molecule.     

Inhibition of SV40 gene expression by microinjected small antisense RNA and DNA molecules

We tested the impact of antisense RNA and DNA molecules on SV40 gene expression by microinjection into TC7 cells. Short antisense stretches, complementary to either hairpin or loop structures on the T antigen mRNA, inhibited T antigen synthesis. In contrast, full-length antisense RNA and DNA molecules did not effect T antigen synthesis.     

A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures.

A technique is described to identify the rare sequences within an RNA molecule that are available for efficient interaction with complementary DNA probes: the target RNA is digested by RNase H in the presence of a random pool of complementary DNA fragments generated from the same DNA preparation that was used for target RNA synthesis. The DNA region was amplified by PCR, partially digested with DNase and denatured prior to RNA binding. In the presence of single-stranded DNA fragments the RNA was digested with RNase H such that, on average, each molecule was cut once. Cleavage sites were detected by gel electrophoresis either directly with end-labeled RNA or by primer extension. The pattern of accessible sites on c- raf mRNA was determined and compared with the known profile of activity of oligonucleotides found in cells, showing the merit of the method for predicting oligonucleotides which are efficient for in vivo antisense targeting. New susceptible sites in the 3'-untranslated region of c- raf mRNA were identified. Also, four RNAs were probed to ascertain to what extent structure predicts accessibility: the P4-P6 domain of the Tetrahymena group I intron, yeast tRNAAsp, Escherichia coli tmRNA and a part of rat 18S rRNA.     

GS介导RNase P对人巨细胞病毒ul54基因mRNA的切割作用

引导序列(Guide Sequences,GSs)是与mRNA靶序列互补并引导RNase P切割的小RNA片段。设计与人巨细胞病毒HCMV(Human Cytomegalovirus,HCMV)ul54基因D片段mRNA序列互补的GS,将其共价结合到大肠杆菌来源RNase P催化核心M1 RNA,构建成T7-M1GS核酶。通过对ul54基因D片段转录产物体外切割实验和将T7-M1GS构建在含有U6启动子的逆转录病毒载体,与构建在真核载体pEGFP-N1的ul54基因D片段共转染人宫颈癌细胞系HeLa的体内切割实验,证实该核酶具备对ul54基因D片段mRNA的特异切割能力,为利用核酶治疗HCMV感染提供实验基础。     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

Inhibition of viral gene expression by human ribonuclease P.

External guide sequences (EGSs) are small RNA molecules which consist of a sequence complementary to a target mRNA and render the target RNA susceptible to degradation by ribonuclease P (RNase P). EGSs were designed to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 for degradation. These EGSs were shown to be able to direct human RNase P to cleave the TK mRNA sequence efficiently in vitro. A reduction of about 80% in the expression level of both TK mRNA and protein was observed in human cells that steadily expressed an EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS which carried a single nucleotide mutation that precluded RNase P recognition. Thus, EGSs may represent novel gene-targeting agents for inhibition of gene expression and antiviral activity.