Small Antisense RNA Research Articles

Prediction of non-coding and antisense RNA genes in Escherichia coli with Gapped Markov Model

A new mathematical index was developed to identify and characterize non-coding RNA (ncRNA) genes encoded within the Escherichia coli ( E. coli) genome. It was designated the GMMI (Gapped Markov Model Index) and used to evaluate sequence patterns located at the separate positions of consensus sequences, codon biases and/or possible RNA structures on the basis of the Markov model. The GMMI was able to separate a set of known mRNA sequences from a mixture of ncRNAs including tRNAs and rRNAs. Consequently, the GMMI was employed to predict novel ncRNA candidates. At the beginning, possible transcription units were extracted from the E. coli genome using consensus sequences for the sigma70 promoter and the rho-independent terminator. Then, these units were evaluated by using the GMMI. This identified 133 candidate ncRNAs, which contain 29 previously annotated small RNA genes and 46 possible antisense ncRNAs. Furthermore 12 transcripts (including five antisense RNAs) were confirmed according to the expression analysis. These data suggests that the expression of small antisense RNAs might be more common than previously thought in the E. coli genome.

Gene silencing of HIV chemokine receptors using ribozymes and single-stranded antisense RNA

The chemokine receptors CXCR4 and CCR5 are required for HIV-1 to enter cells, and the progression of HIV-1 infection to AIDS involves a switch in the co-receptor usage of the virus from CCR5 to CXCR4. These receptors therefore make attractive candidates for therapeutic intervention, and we have investigated the silencing of their genes by using ribozymes and single-stranded antisense RNAs. In the present study, we demonstrate using ribozymes that a depletion of CXCR4 and CCR5 mRNAs can be achieved simultaneously in human PBMCs (peripheral blood mononuclear cells), cells commonly used by the virus for infection and replication. Ribozyme activity leads to an inhibition of the cell-surface expression of both CCR5 and CXCR4, resulting in a significant inhibition of HIV-1 replication when PBMCs are challenged with the virus. In addition, we show that small single-stranded antisense RNAs can also be used to silence CCR5 and CXCR4 genes when delivered to PBMCs. This silencing is caused by selective degradation of receptor mRNAs.

Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression.

Stable Triplet of Uracil−Uracil Basepairs in a Small Antisense RNA

The interaction between ant (antirepressor) mRNA and its antisense RNA, sar (small antisense RNA), is important in regulation of the development of bacteriophage P22. Sar is 68-69 nucleotides in length and is believed to consist of two hairpin structures separated by a small inter-hairpin region, followed by a short 3' tail [Schaefer and McClure RNA 1997, 3, 141-156]. A novel feature of the proposed secondary structure of the first hairpin is a extremely rare triple U:U base stack. Heteronuclear NMR studies presented here show that the first hairpin does not possess a unique structure in the absence of the second hairpin. However, it acquires a well-defined structure within full length sar. Remarkably, the triple U:U stack appears to be a stable feature of the first hairpin, regardless of the presence or absence of the second hairpin.

RNase E-based ribonucleoprotein complexes: mechanical basis of mRNA destabilization mediated by bacterial noncoding RNAs

Hfq-binding antisense small RNAs of Escherichia coli, SgrS and RyhB, mediate the destabilization of target mRNAs in an RNase E-dependent manner. SgrS, whose expression is induced in response to phosphosugar stress, act on the ptsG mRNA encoding a major glucose transporter, while RyhB, whose expression is induced in response to Fe depletion, acts on several mRNAs encoding Fe-binding proteins. In this report, we addressed the question of how SgrS and RyhB RNAs cooperate with RNase E to destabilize the target mRNAs. We demonstrate that Hfq along with SgrS and RyhB copurified with RNase E but not with truncated RNase E. In addition, we show that RNase E but not other degradosome components copurified with Hfq. Taken together, we conclude that RNase E forms variable ribonucleoprotein complexes with Hfq/small RNAs through its C-terminal scaffold region. These complexes, distinct from the RNA degradosome, may act as specialized RNA decay machines that initiate the degradation of mRNAs targeted by each small RNA. The present finding has uncovered the mechanical basis of mRNA destabilization mediated by bacterial small RNAs. The formation of ribonucleoprotein complexes containing RNases could be a general way by which small RNAs destabilize target mRNAs in both prokaryotes and eukaryotes.

Small Sense and Antisense RNAs Derived from a Telomeric Retroposon Family in Giardia intestinalis

Sequencing of a library of small RNAs from Giardia intestinalis identified a novel class of small sense and antisense RNAs homologous to the retroposon family GilT/Genie1 that is located at certain telomeres. These small RNAs may contribute to silencing GilT expression via the RNA interference pathway.

Initiation of Protein Synthesis in Bacteria

Valuable information on translation initiation is available from biochemical data and recently solved structures. We present a detailed description of current knowledge about the structure, function, and interactions of the individual components involved in bacterial translation initiation. The first section describes the ribosomal features relevant to the initiation process. Subsequent sections describe the structure, function, and interactions of the mRNA, the initiator tRNA, and the initiation factors IF1, IF2, and IF3. Finally, we provide an overview of mechanisms of regulation of the translation initiation event. Translation occurs on ribonucleoprotein complexes called ribosomes. The ribosome is composed of a large subunit and a small subunit that hold the activities of peptidyltransfer and decode the triplet code of the mRNA, respectively. Translation initiation is promoted by IF1, IF2, and IF3, which mediate base pairing of the initiator tRNA anticodon to the mRNA initiation codon located in the ribosomal P-site. The mechanism of translation initiation differs for canonical and leaderless mRNAs, since the latter is dependent on the relative level of the initiation factors. Regulation of translation occurs primarily in the initiation phase. Secondary structures at the mRNA ribosomal binding site (RBS) inhibit translation initiation. The accessibility of the RBS is regulated by temperature and binding of small metabolites, proteins, or antisense RNAs. The future challenge is to obtain atomic-resolution structures of complete initiation complexes in order to understand the mechanism of translation initiation in molecular detail.

Implication of membrane localization of target mRNA in the action of a small RNA: mechanism of post-transcriptional regulation of glucose transporter in Escherichia coli

Accumulation of phosphosugars such as glucose-6-phosphate causes a rapid degradation of ptsG mRNA encoding the major glucose transporter IICB(Glc) in an RNase E/degradosome-dependent manner. The destabilization of ptsG mRNA is caused by a small antisense RNA (SgrS) that is induced by phosphosugar stress. In this study, we analyzed a series of ptsG-crp translational fusions to identify the mRNA region required for the rapid degradation of ptsG mRNA. We found that the ptsG-crp mRNA is destabilized in response to phosphosugar stress when it contains the 5' portion of ptsG mRNA corresponding up to the first two transmembrane domains (TM1 and TM2) of IICB(Glc). The destabilization of ptsG-crp mRNA was largely eliminated by frameshift mutations in the transmembrane region. The IICB(Glc)-CRP fusion proteins containing more than two transmembrane domains were localized at the membrane. The efficient destabilization of ptsG-crp mRNA was restored when TM1 and TM2 of IICB(Glc) were replaced by part of the LacY transmembrane region. We conclude that the membrane-targeting property of IICB(Glc) protein rather than the particular nucleotide or amino acid sequence is required for the efficient degradation of ptsG mRNA in response to metabolic stress. The stimulation of ptsG-crp mRNA degradation was completely eliminated when either the hfq or sgrS gene is inactivated. The efficient mRNA destabilization was observed in the absence of membrane localization when translation was reduced by introducing a mutation in the ribosome-binding site in the cytoplasmic ptsG-crp mRNA. Taken together, we conclude that mRNA localization to the inner membrane coupled with the membrane insertion of nascent peptide mediates the Hfq/SgrS-dependent ptsG mRNA destabilization presumably by reducing second rounds of translation.

Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids

Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors.

Two discrete elements are required for the replication of a repABC plasmid: an antisense RNA and a stem–loop structure

The repABC replicons contain an operon encoding the initiator protein (RepC) and partitioning proteins (RepA and RepB). The latter two proteins negatively regulate the transcription of the operon. In this article we have identified two novel regulatory elements, located within the conserved repB-repC intergenic sequence, which negatively modulate the expression of repC, in plasmid p42d of Rhizobium etli. One of them is a small antisense RNA and the other is a stem-loop structure in the repABC mRNA that occludes the Shine-Dalgarno sequence of repC. According to in vivo and in vitro analyses, the small antisense RNA (57-59 nt) resembles canonical negative regulators of replication because: (i) it is transcribed from a strong constitutive promoter (P2), (ii) the transcript overlaps untranslated region upstream of the RepC coding sequences, (iii) the RNA forms one secondary structure acting as a rho-independent terminator, (iv) the antisense RNA is a strong trans-incompatibility factor and (v) its presence reduces the level of repC expression. Surprisingly, both of these seemingly negative regulators are required for efficient plasmid replication.

Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells

Introns are present in some human pre-tRNAs. They are spliced out during the maturation processes of pre-tRNAs in a way that is irrelevant to their specific nucleotide sequences. This unique characteristic of tRNA splicing can be used for generation of small antisense RNAs by replacing the intron sequences with corresponding antisense sequences. In this work, the intron sequence of human pre-tRNA tyr gene was replaced with a 20 bp antisense sequence targeted to the 5 ′ coding region of cyclin D1, a molecule that was over-expressed in many malignant proliferating cells. Under the control of U6 SnRNA promoter to further enhance transcription efficiency of the modified pre-tRNA tyr gene and subsequent antisense generation, the antisense RNA exhibited obvious suppression of cyclin D1 expression in H22 hepatoma cells. The growth of H22-transplanted tumors in mice was significantly inhibited when treated with naked plasmid DNA harboring the cyclin D1 antisense RNA generating cassette. Such tumor growth inhibition might be due to apoptosis caused by reduced cyclin D1 expression as revealed by immunohistochemical analysis of tumor samples.

Degradation of targeted mRNAs in Escherichia coli: regulation by a small antisense RNA.

In this issue of Genes & Development, Masse et al. (2003a) show that a small antisense RNA (saRNA) triggers mRNA degradation via a pathway in which the saRNA is also consumed. The stoichiometric binding of the saRNA to its mRNA target thus appears to activate the degradation of both RNAs (coupled degradation). The physical interaction of complementary polynucleotides is arguably the fundamental chemical principle underlying molecular genetics. It is the most important feature of the structure of double-stranded DNA, and it is at the heart of the mechanism by which tRNA decodes mRNA to synthesize protein. The article by Masse et al. (2003a) reports novel findings on the action of a chromosome-encoded saRNA in the regulation of gene expression. They show that RyhB, an saRNA, triggers mRNA degradation via a pathway in which RyhB is degraded together with its target mRNA. Similar results were obtained with two other saRNAs. Thus, it seems likely that RyhB will serve as a model for saRNAs that activate mRNA degradation. Biological regulatory mechanisms involving antisense RNA have been recognized for many years in Escherichia coli. However, until recently, the known antisense RNAs were encoded by extrachromosomal elements such as transposons, plasmids, and bacteriophage. With the current renaissance in interest in small noncoding RNAs, a surprising number of putative small regulatory RNAs (50–100), encoded on the E. coli chromosome, have recently been described. An important factor in the discovery of these RNAs was the availability of the complete genome sequence of E. coli and related bacteria such as Salmonella. This genomic information facilitated searches based, in part, on sequence conservation in noncoding regions of the chromosome. It is only now that the function of some of the newly discovered RNAs is being elucidated. Although it seems reasonable to believe that the chromosome-encoded saRNAs and the classical antisense RNAs from transposons, plasmids, and bacteriophage act by similar mechanisms, I will argue that there could be some significant differences. This perspective is based on the accompanying research article by Masse et al. (2003a). As a reference for recent developments on small regulatory RNAs in E. coli, I highly recommend the review by Susan Gottesman that was published in Genes & Development last year (Gottesman 2002). In this perspective, I will describe the antisense RNAs from transposons, plasmids, and bacteriophage that have been characterized over the past twenty years (classical antisense), then introduce the chromosome-encoded antisense RNAs that have been discovered recently (renaissance antisense). After that, I will briefly discuss some differences between these two families of antisense RNA, then discuss the work in the Masse et al. (2003a) article and its implications for the role of the chromosome-encoded antisense RNAs in mRNA degradation.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

Proteolysis in prokaryotes: protein quality control and regulatory principles.

Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes.

Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M. circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M. circinelloides a suitable organism for investigating some unresolved questions in RNA silencing.

Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli.

RyhB is a small antisense regulatory RNA that is repressed by the Fur repressor and negatively regulates at least six mRNAs encoding Fe-binding or Fe-storage proteins in Escherichia coli. When Fe is limiting, RyhB levels rise, and target mRNAs are rapidly degraded. RyhB is very stable when measured after treatment of cells with the transcription inhibitor rifampicin, but is unstable when overall mRNA transcription continues. We propose that RyhB turnover is coupled to and dependent on pairing with the target mRNAs. Degradation of both mRNA targets and RyhB is dependent on RNase E and is slowed in degradosome mutants. RyhB requires the RNA chaperone Hfq. In the absence of Hfq, RyhB is unstable, even when general transcription is inhibited; degradation is dependent upon RNase E. Hfq and RNase E bind similar sites on the RNA; pairing may allow loss of Hfq and access by RNase E. Two other Hfq-dependent small RNAs, DsrA and OxyS, are also stable when overall transcription is off, and unstable when it is not, suggesting that they, too, are degraded when their target mRNAs are available for pairing. Thus, this large class of regulatory RNAs share an unexpected intrinsic mechanism for shutting off their action.

The phage lambda CII transcriptional activator carries a C-terminal domain signaling for rapid proteolysis.

ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the lambda CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the lambda CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage lambda carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein.

Pseudoknot-dependent translational coupling in repBA genes of the IncB plasmid pMU720 involves reinitiation.

Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

The influence of inverted repeats on the production of small antisense RNAs involved in gene silencing.

Gene silencing by an ACO1 [1-aminocyclopropane-1-carboxylate (ACC) oxidase 1] sense transgene in tomato plants was correlated with the production of small antisense RNAs (asRNAs) of 21-28 nucleotides, which were preferentially generated from the 3' region of the transgene. Adding inverted repeats (IRs) to the 5' untranslated region of the ACO1 transgene led to stronger silencing than was obtained with the transgene lacking the IRs, and in these plants the asRNAs were preferentially produced from the 5' region, including the IRs themselves and sequences immediately downstream. This observation indicates that secondary structure, including inverted repeats, may be a key determinant of small RNA production in gene silencing. Small asRNAs of 28 nt were much more abundant in the line containing the IRs than in the line without IRs, and may contribute to the stronger silencing associated with the IRs. Much lower levels of small RNA species were detected in plants containing an antisense ACO1 transgene than in an ACO1-sense silenced line showing weaker silencing. This suggests that the stronger suppression of the endogenous ACO1 gene by an antisense transgene may be the result of the combined effects of large antisense RNAs produced from the antisense transgene and small asRNAs.

Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora.

Small RNA molecules have been found to be specifically associated with posttranscriptional gene silencing (PTGS) in both plants and animals. Here, we find that small sense and antisense RNAs are also involved in PTGS in Neurospora crassa. The accumulation of these RNA molecules depends on the presence of functional qde-1 and qde-3 genes previously shown to be essential for gene silencing, but does not depend on a functional qde-2, indicating that this gene is involved in a downstream step of the gene silencing pathway. Supporting this idea, a purified QDE2 protein complex was found to contain small RNA molecules, suggesting that QDE2 could be part of a small RNA-directed ribonuclease complex involved in sequence-specific mRNA degradation.

Relationship between small antisense RNAs and aberrant RNAs associated with sense transgene mediated gene silencing in tomato.

We investigated the occurrence and properties of small sense and antisense RNAs and aberrant transcripts in transgenic tomato plants exhibiting post-transcriptional silencing of the endogenous polygalacturonase (PG) sense gene and a truncated homologous transgene. Small antisense RNAs, about 23 nucleotides in size, were present in the leaves and immature green fruit where the transgene was transcribed in the absence of transcripts from the fruit ripening-specific endogenous PG gene. This suggests that the small antisense RNAs were produced from the transgene, which was supported by their failure to hybridise with the 3' half of the endogenous PG gene, the region that was excluded from the transgene. The features of two aberrant RNAs, corresponding to the 5' and 3' halves of the endogenous gene, strongly suggest that they arise by endonucleolytic cleavage of endogenous PG mRNA. The region where cleavage occurs is associated with the preferential production of small RNAs from the 3' end of the transgene. These results suggest a model where small antisense RNAs generated from the 3' end of the transgene anneal to the endogenous mRNA, resulting in cleavage at or near the annealing regions.