Promoter cross-talk affects the inducible expression of intronic shRNAs from the tetracycline response element.

BACKGROUND: RNA interference (RNAi), defined as double-stranded, RNA-mediated gene silencing, is a useful tool for functional genomic studies. Along with increasing information about genomic sequences due to the innovative development of genome-sequencing technologies, functional genomic technologies are needed to annotate the genome and determine the processes by which each gene is regulated. Lentiviral vectors have been used to efficiently deliver reagents, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), into cells and tissues for functional genomic analyses. OBJECTIVE: We developed a lentiviral vector that efficiently expresses intronic shRNA from the tetracycline regulatory element (TRE) promoter in a doxycycline-dependent manner. METHODS: We developed a lentiviral vector system that contains reverse tetracycline-controlled transactivator 3 (rtTA3) and the TRE promoter, which are necessary for the doxycycline-inducible expression of shRNAs that are expressed as intronic miR-30a precursors. We then measured the cross-talk between the cytomegalovirus (CMV) and TRE promoters in the vector. RESULTS: We found that nearby promoters influence each other and that the TRE promoter should be located far from other promoters, such as the CMV promoter, in a vector. The orientation of a promoter with respect to other promoters also influences its transcriptional activity. A head-to-head orientation of the CMV and TRE promoters maintains the lowest level of transcription from TRE in the absence of doxycycline, compared to the tail-to-tail and head-to-tail orientations. CONCLUSION: Based on these findings, we were able to construct a lentiviral vector that faithfully expresses intronic miR-30a shRNA precursors in a doxycycline-inducible manner.

Enhancement of gene knockdown efficiency by CNNC motifs in the intronic shRNA precursor.

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Of several methods to express shRNA, a method of transcribing shRNAs embedded in microRNA precursors has been more widely used than the one that directly expresses shRNA from RNA polymerase III promoters because the microRNA precursor form of shRNA is known to cause lower levels of cytotoxicity and off-target effects than the overexpressed shRNAs from the RNA polymerase III promoters. OBJECTIVE: We study the primary sequence features of microRNA precursors, which enhance their processing into mature form, helps design more potent shRNA precursors embedded in microRNA precursors. METHODS: We measure the enhancement of gene knockdown efficiency by adding CNNC motifs in the 3' flanking region of shRNA precursor embedded in the human miR-30a microRNA precursor. RESULTS: By systemically adding three CNNC motifs in the 3' flanking region of shRNA precursor, we found that addition of two CNNC motifs saturates their enhanced knockdown ability of shRNA and that the CNNC motif in the + 17 to + 20 from the drosha cleavage site is most important for the shRNA-mediated target gene knock down. We also did see little knockdown of target gene expression by the shRNA precursor lacking CNNC motif. CONCLUSION: Since genetic studies generally require techniques that could reduce gene expression at different degrees, the findings in this study will allow us to use RNAi for genetic studies of reducing gene expression at different degrees.

Enzymatic construction of shRNA library from oligonucleotide library.

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. OBJECTIVE: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. METHODS: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. RESULTS: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. CONCLUSION: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.

LIN-12/Notch regulates lag-1 and lin-12 expression during anchor cell/ventral uterine precursor cell fate specification.

During Caenorhabditis elegans gonadal development, a stochastic interaction between the LIN-12/Notch receptor and the LAG-2/Delta ligand initiates cell fate specification of two equivalent pre-anchor cell (AC)/pre-ventral uterine (VU) precursor cells. Both cells express lin-12 and lag-2 before specification, and a small difference in LIN-12 activity leads to the exclusive expression of lin-12 in VUs and lag-2 in the AC through an unknown feedback mechanism. In this Notch signaling process, the cleaved LIN-12/Notch intracellular domain (NICD) binds to the LAG-1/CSL transcriptional repressor, forming a transcriptional activator complex containing LAG-1 and NICD. Here we show that clustered LAG-1 binding sites in lin-12 and lag-1 are involved in regulating lin-12 and lag-1 expression during AC/VU cell fate specification. Both genes are expressed in VU cells, but not the AC, after specification. We also show that lin-12 is necessary for lag-1 expression in VU cells. Interestingly, lin-12 (null) animals express lag-1 in the AC, suggesting that LIN-12 signaling is necessary for the suppression of lag-1 expression in the AC. Ectopic expression of lag-1 cDNA in the AC causes a defect in the vulvaluterine (V-U) connection; therefore, LAG-1 should be eliminated in the AC to form a normal V-U connection at a later developmental stage in wild-type animals.

Clustered LAG-1 binding sites in lag-1/CSL are involved in regulating lag-1 expression during lin-12/Notch-dependent cell-fate specification.

The cell-fate specification of the anchor cell (AC) and a ventral uterine precursor cell (VU) in Caenorhabditis elegans is initiated by a stochastic interaction between LIN-12/Notch receptor and LAG-2/Delta ligand in two neighboring Z1.ppp and Z4.aaa cells. Both cells express lin-12 and lag-2 before specification, and a small difference in LIN-12 activity leads to the exclusive expressions of lin-12 in VU and lag-2 in the AC, through a feedback mechanism of unknown nature. Here we show that the expression pattern of lag-1/CSL, a transcriptional repressor itself that turns into an activator upon binding of the intracellular domain of Notch, overlaps with that of lin-12. Site-directed mutagenesis of LAG-1 binding sites in lag-1 maintains its expression in the AC, and eliminates it in the VU. Thus, AC/VU cell-fate specification appears to involve direct regulation of lag-1 expression by the LAG-1 protein, activating its transcription in VU cells, but repressing it in the AC.