Intersection of Small RNA Pathways in Arabidopsis thaliana Sub-Nuclear Domains

In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.     

Masked antisense: a molecular configuration for discriminating similar RNA targets

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These ‘masked’ antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR–ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.     

Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis

A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10⁴Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences.     

The Essential Function of B. subtilis RNase III Is to Silence Foreign Toxin Genes

RNase III–related enzymes play key roles in cleaving double-stranded RNA in many biological systems. Among the best-known are RNase III itself, involved in ribosomal RNA maturation and mRNA turnover in bacteria, and Drosha and Dicer, which play critical roles in the production of micro (mi)–RNAs and small interfering (si)–RNAs in eukaryotes. Although RNase III has important cellular functions in bacteria, its gene is generally not essential, with the remarkable exception of that of Bacillus subtilis. Here we show that the essential role of RNase III in this organism is to protect it from the expression of toxin genes borne by two prophages, Skin and SPβ, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, B. subtilis requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify txpA and yonT as the two toxin-encoding mRNAs of Skin and SPβ that are sensitive to RNase III. We further explore the mechanism of RNase III–mediated decay of the txpA mRNA when paired to its antisense RNA RatA, both in vivo and in vitro.     

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]     

Enzymatic production and expression of shRNAmir30 from cDNAs

RNA interference (RNAi) is an important tool for studying gene function and genetic networks. Double-stranded RNA (dsRNA) triggers RNAi that selectively silences gene expression mainly by degrading target mRNA sequences. Short interfering RNA, short hairpin RNA (shRNA), long dsRNA, and microRNA-based shRNA (shRNAmir) are four different types of dsRNA that have been widely used to silence gene expression in cultured cells, tissues, organs, and organisms. Long dsRNAs are usually 200–500 nucleotides in length and can selectively suppress expression of target genes in Caenorhabditis elegans and Drosophila but not in mammals due to unwanted non-specific knockdown. Thus, multiple attempts have been made to synthesize, express, and deliver short dsRNAs that specifically silence target genes in mammals. We describe a method for constructing an RNAi library by converting cDNAs into shRNAmir30 sequences by sequential treatment with different enzymes and affinity purification of biotin- or digoxygenin-labeled DNA fragments. We also developed a system to generate stable cell lines that uniformly express shRNAmir30s and fluorescence reporters by Cre recombinase-dependent site-specific recombination. Thus, combined with the RNAi library, this system facilitates screening for potent RNAi sequences that strongly suppress expression of target genes.