Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location

The internal ribosomal entry site (IRES)from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Compared head-to-head with dual-luciferase reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a similarly configured pIRES vector. It also produced nearly twice as much protein as pCITE-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. The preferred IRES (viral bases 273-845) and the minimum IRES (viral bases 400-836) for optimum activity are illustrated.     

Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase

Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P(T7)) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P(T7)-driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene alpha-tubulin locus. A plasmid construct with P(T7) and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the non-coding strand of an 18SrRNA gene, resulted in tetracycline-inducible expression of the reporter as much as 16- and 150-fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P(T7) augmented the reporter's expression 2-fold more in comparison to P(T7)-less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.     

RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase

Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.     

Human cell lines expressing hormone regulated T7 RNA polymerase localized at distinct intranuclear sites

Although several systems are now available for the controlled expression of eukaryotic genes transcribed by RNA polymerase II, regulated expression has been more difficult to achieve in the case of genes transcribed by RNA polymerase III. In the present study the gene for bacteriophage T7 RNA polymerase, implanted with a eukaryotic nuclear localization signal, was linked to a 5'-flanking ecdysone-responsive promoter and stably transformed human cell lines were constructed in which the ecdysone promoter-T7 RNA polymerase gene had been integrated intact, as demonstrated by a polymerase chain reaction assay. Exposure of these cells to the ecdysone analog ponasterone A resulted in the appearance of a single protein having the expected size of T7 RNA polymerase in immunoblots of cell extracts probed with an affinity purified antibody raised against the C-terminus of T7 RNA polymerase. The induced T7 RNA polymerase was exclusively localized in the nucleus of induced cells and was undetectable in uninduced cells either by immunoblotting or immunofluorescence. The induced T7 RNA polymerase was present at numerous punctate foci dispersed throughout the nucleoplasmic regions of the nucleus and was also present in the nucleoli. Both of these observed intranuclear localizations have relevance to the potential applications of this system.     

DNA sequence for the T7 RNA polymerase promoter for T7 RNA species II

The DNA sequence for the T7 late region class III promoter for T7 RNA species II has been determined. I have found that the DNA sequence for this promoter presented in an earlier report (Oakley et al., 1979) is incorrect and that this class III promoter contains a 23 base-pair sequence identical to those present in all other T7 class III promoters (Rosa, 1979). The T7 RNA species II promoter has been located at 68% on the T7 genome.     

A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10⁵ copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.     

A novel T7 system utilizing mRNA coding for T7 RNA polymerase

The T7 system dose not require the relocation of a reporter gene to the nucleus for its gene expression in the cytoplasm, but relies on the co-localization of T7 RNA polymerase (T7 RNAP) enzyme and reporter gene DNA that is controlled by the T7 promoter. In the present study, we developed a new T7 system in that gene expression can occur at a higher level than those using conventional systems. Insertion of 5'- and 3'-untranslated regions (UTR) of beta-globin gene into a reporter gene enhanced the reporter gene expression, presumably due to the stability and efficient translation of the mRNA. Instead of the T7 RNAP protein used in conventional methods, moreover, transfection of cells with T7 RNAP mRNA, which has been modified by inserting beta-globin 5'- and 3'-UTR sequences as well as the cap and poly(A) tail structures, further enhanced the reporter gene expression. Thus, this novel T7 system using T7 RNAP mRNA may be powerful for the efficient gene expression of DNA exogenously provided in the cytoplasm.     

Strong suppression of chloramphenicol acetyltransferase expression by inducing T7 RNA polymerase

Summary Suppression of gene expression can be used as a way to study gene regulation. Expression of chloramphenicol acetyltransferase (CAT) underE. coli promoter was inhibited by 92 % by induction of T7 RNA polymerase which transcribes from a T7 promoter opposingE. coli promoter.