RNA In Mammalian Cells Research Articles

The Puzzle of RNAs that Target Gene Promoters

Setting the right target: Most researchers who use small RNAs in mammalian cells assume that mRNA will be the target. Recent studies suggest that small RNAs can also target chromosomal DNA. Few discoveries have had the impact of RNAi. Most researchers who use small RNAs to control gene expression in mammalian cells assume that mRNA will be the target. Recent studies, however, have suggested that small RNAs can also target chromosomal DNA.

Use of Bombyx mori U6 promoter for inducing gene-silencing in silkworm cells

Double-stranded (ds) RNA is a useful tool for sequence-specific gene silencing, now known as RNA interfer ence (RNAi), in a variety of organisms. Studies on pol III promoter-based expression systems have been well documented showing them to be an effective tool for inducing RNAi in mammalian cells and Drosophila Schneider 2 (S2) cells, but not in silkworm cells. In this study, we developed a Bombyx mori U6 promoter (U6p)based short hairpin RNA (shRNA) expression system for inducing RNAi in silkworm cells. First, a transient reporter assay showed that the transfection of plasmids containing U6p-driven shRNA-expression units (U6-shRNA units) caused a decrease in the expression of the target gene in BmN cells with the efficacy dependent on the length of the hairpin stem region. Subsequently, we demonstrated the ability of the U6-shRNA unit to confer resistance to Bombyx mori nucleopolyhedrovirus (BmNPV)-resistance on BmN cells; i.e., the viral titer in the culture medium of BmN cells transfected with U6-shRNA (with the sequence of an essential viral gene)-expression plasmids was decreased to 30% of the control at 48 hr post-infection. These results suggested that the U6shRNA expression system was a useful tool for RNAi in silkworm cells.

Biological basis for restriction of microRNA targets to the 3′ untranslated region in mammalian mRNAs

MicroRNAs (miRNAs) interact with target sites located in 3' untranslated regions (3'UTR) of mRNAs to down-regulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target localization in the 3' UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as siRNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs, and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

Self-assembly of sapovirus recombinant virus-like particles from polyprotein in mammalian cells

The SaV genome is a positive-sense, non-segmented single-strand RNA molecule of approximately 7.5 kb that is polyadenylated at its 3' terminus. The major capsid (VP1) of SaV is thought to be produced as the ORF1 polyprotein followed by cleavage, or translation from subgenomic RNA (3'-coterminal with the virus genome), or both. We have recently reported the formation of SaV VLP from subgenomic-like RNA in mammalian cells. In the present study, we demonstrated that the VP1 cleaved from a part of ORF1 polyprotein self-assembled into VLP in mammalian cells when a transient expression system using a recombinant vaccinia virus encoding T7 RNA polymerase was used.

Human Telomeric RNA in G-quadruplex Structure

Very recently, a breaking finding from two groups demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA in mammalian cells. Telomeric RNA, a newly appeared player in telomere biology, may be a key component of telomere machinery. However, structure and function of the telomeric RNA in chromosome ends have not yet been elucidated. In the current studies, we found that the human telomeric RNA sequence can form parallel G-quadruplex structure in the presence of K(+) or Na(+) ions. These results provide valuable information to allow understanding of the roles of human telomeric RNA in chromosome ends regulation and protection.

G-Quadruplex Formation by Human Telomeric Repeats-Containing RNA in Na+ Solution

For a long time, telomeres have been considered to be transcriptionally silent. Very recently, a breaking finding from two groups demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA in mammalian cells (Azzalin, C. M.; Reichenbach, P.; Khoriauli, L.; Giulotto, E.; Lingner, J. Science 2007, 318, 798-801. Schoefter, S.; Blasco, M. A. Nat. Cell Biol. 2008, 10, 228-236). The telomeric RNA, a newly appeared player in telomere biology, may be a key component of telomere machinery. In the current study, we used a combination of NMR, circular dichroism (CD), matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS), and gel electrophoresis to investigate the structural features of a human telomere RNA sequence. We demonstrated that human telomere RNA can form a parallel G-quadruplex structure in the presence of Na(+). Importantly, we found for the first time that the G-quadruplex forming telomere RNA protects itself from enzymatic digestion. These results provide valuable information to allow understanding of the structure and function of human telomeric RNA.

The Efficacy of Generating Three Independent Anti-HIV-1 siRNAs from a Single U6 RNA Pol III-Expressed Long Hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.

Genome-wide screen reveals APC-associated RNAs enriched in cell protrusions

RNA localization is important for the establishment and maintenance of polarity in multiple cell types. Localized RNAs are usually transported along microtubules or actin filaments and become anchored at their destination to some underlying subcellular structure. Retention commonly involves actin or actin-associated proteins, although cytokeratin filaments and dynein anchor certain RNAs. RNA localization is important for diverse processes ranging from cell fate determination to synaptic plasticity; however, so far there have been few comprehensive studies of localized RNAs in mammalian cells. Here we have addressed this issue, focusing on migrating fibroblasts that polarize to form a leading edge and a tail in a process that involves asymmetric distribution of RNAs. We used a fractionation scheme combined with microarrays to identify, on a genome-wide scale, RNAs that localize in protruding pseudopodia of mouse fibroblasts in response to migratory stimuli. We find that a diverse group of RNAs accumulates in such pseudopodial protrusions. Through their 3' untranslated regions these transcripts are anchored in granules concentrated at the plus ends of detyrosinated microtubules. RNAs in the granules associate with the adenomatous polyposis coli (APC) tumour suppressor and the fragile X mental retardation protein (FMRP). APC is required for the accumulation of transcripts in protrusions. Our results suggest a new type of RNA anchoring mechanism as well as a new, unanticipated function for APC in localizing RNAs.

Expression and functional analyses of PCA‐1 (ABH3) for molecular‐targeted therapy of prostate cancer

Prostate cancer is the most common malignancy in men and the second leading cause of cancer death in Western countries. Many studies found out a number of genetic changes in prostate tumorigenesis, but it is not sufficient to understand the molecular mechanism of prostate tumorigenesis. We have recently identified and cloned a new gene named prostate cancer antigen‐1 (PCA‐1), which shows higher expression of both mRNA and protein in prostate cancer than in normal prostate tissue. Database analysis indicated that PCA‐1 has high similarity to Escherichia coli AlkB, a DNA and RNA alkylation damage repair enzyme, and belongs to the ABH (AlkB homologue) family as ABH3. PCA‐1 localized in cytoplasm as well as nuclei in some specimens of prostate cancer and in transfected HeLa cells, supporting that PCA‐1 might repair alkylated DNA and RNA in mammalian cells. To investigate the function of PCA‐1, we used the RNA interference technique to specifically deplete PCA‐1 mRNA in the prostate cancer cell lines DU145 and PC3. Transfection with PCA‐1 siRNAs causeda dramatic reduction in PCA‐1 at both mRNA and protein levels (to less than 20% of the levelsseen in control siRNA transfectants for 48 h culture). Interestingly, down‐regulation of PCA‐1 by siRNAs markedly inhibited cell proliferation and induced cell death by apoptosis. These results suggest that PCA‐1 may be a new molecular‐target for the treatment of human prostate cancer.

Invertebrate 7SK snRNAs

7SK RNA is a highly abundant noncoding RNA in mammalian cells whose function in transcriptional regulation has only recently been elucidated. Despite its highly conserved sequence throughout vertebrates, all attempts to discover 7SK RNA homologues in invertebrate species have failed so far. Here we report on a combined experimental and computational survey that succeeded in discovering 7SK RNAs in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. Despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. The additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3′ and 5′ stem-loop motifs, and provide evidence for a third, structurally well-conserved domain.

An efficient RNA‐cleaving DNA enzyme can specifically target the 5′‐untranslated region of severe acute respiratory syndrome associated coronavirus (SARS‐CoV)

BackgroundThe worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS‐CoV. We report the use of DNAzyme (catalytic DNA) to target the 5′‐untranslated region (5′UTR) of a highly conserved fragment in the SARS genome as an approach to suppression of SARS‐CoV replication. A mono‐DNA enzyme (Dz‐104) possessing the 10–23 catalytic motif was synthesized and tested both in vitro and in cell culture.Materials and methodsSARS‐CoV total RNA was isolated, extracted from the SARS‐CoV‐WHU strain and converted into cDNA. We designed a RNA‐cleaving 10–23 DNAzyme targeting at the loop region of the 5′UTR of SARS‐CoV. The designed DNAzyme, Dz‐104, and its mutant version, Dz‐104 (mut), as a control consist of 9 + 9 arm sequences with a 10–23 catalytic core. In vitro cleavage was performed using an in vitro transcribed 5′UTR RNA substrate. A vector containing a fused 5′UTR and enhanced green fluorescent protein (eGFP) was co‐transfected with the DNAzyme into E6 cells and the cells expressing eGFP were visualized with fluorescence microscopy and analyzed by fluorescence‐activated cell sorting (FACS).Results and conclusionsOur results demonstrated that this DNAzyme could efficiently cleave the SARS‐CoV RNA substrate in vitro and inhibit the expression of the SARS‐CoV 5′UTR‐eGFP fusion RNA in mammalian cells. This work presents a model system to rapidly screen effective DNAzymes targeting SARS and provides a basis for potential therapeutic use of DNA enzymes to combat the SARS infection. Copyright © 2007 John Wiley & Sons, Ltd.

MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells

MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.

Characterization of the variable region in the 3′ non-translated region of dengue type 1 virus

The first 84 nt in the 3' non-translated region (3' NTR) of dengue type 1 virus (DENV-1) exhibit lower levels of conservation than the other regions; this region is named the variable region (VR). The VR is further divided into two subregions: a 5'-terminal hypervariable region (HVR) and a 3'-terminal semi-variable region (SVR). Recent reports suggested that the VR of DENV-2 is required for efficient virus growth in mammalian cells. To investigate whether this is also true for the VR of DENV-1, deletion or replacement mutations were introduced into the VR by using recombinant DENV-1 cDNA clones. Recombinant viruses with deletion of either or both subregions exhibited reduced growth properties compared with the original virus. Mutants with incompletely reversed or unrelated sequences in the HVR demonstrated growth properties similar to those of the original virus. However, a replacement mutation in the SVR did not cause recovery of growth properties. Furthermore, the amount of viral RNA was decreased in Vero cells infected with the growth-attenuated mutant viruses. Results of reporter translation assays suggest that VR mutations may not affect the translation process of DENV-1. These data indicate that the VR is important for DENV-1 replication and is associated with the accumulation of DENV-1 RNA in mammalian cells, and that the HVR and SVR in the VR may have different roles in DENV-1 replication.

Lentiviral vectors encoding tetracycline-dependent repressors and transactivators for reversible knockdown of gene expression: a comparative study

BackgroundRNA interference (RNAi)-mediated by the expression of short hairpin RNAs (shRNAs) has emerged as a powerful experimental tool for reverse genetic studies in mammalian cells. A number of recent reports have described approaches allowing regulated production of shRNAs based on modified RNA polymerase II (Pol II) or RNA polymerase III (Pol III) promoters, controlled by drug-responsive transactivators or repressors such as tetracycline (Tet)-dependent transactivators and repressors. However, the usefulness of these approaches is often times limited, caused by inefficient delivery and/or expression of shRNA-encoding sequences in target cells and/or poor design of shRNAs sequences. With a view toward optimizing Tet-regulated shRNA expression in mammalian cells, we compared the capacity of a variety of hybrid Pol III promoters to express short shRNAs in target cells following lentivirus-mediated delivery of shRNA-encoding cassettes.ResultsRNAi-mediated knockdown of gene expression in target cells, controlled by a modified Tet-repressor (TetR) in the presence of doxycycline (Dox) was robust. Expression of shRNAs from engineered human U6 (hU6) promoters containing a single tetracycline operator (TO) sequence between the proximal sequence element (PSE) and the TATA box, or an improved second-generation Tet-responsive promoter element (TRE) placed upstream of the promoter was tight and reversible as judged using quantitative protein measurements. We also established and tested a novel hU6 promoter system in which the distal sequence element (DSE) of the hU6 promoter was replaced with a second-generation TRE. In this system, positive regulation of shRNA production is mediated by novel Tet-dependent transactivators bearing transactivation domains derived from the human Sp1 transcription factor.ConclusionOur modified lentiviral vector system resulted in tight and reversible knockdown of target gene expression in unsorted cell populations. Tightly regulated target gene knockdown was observed with vectors containing either a single TO sequence or a second-generation TRE using carefully controlled transduction conditions. We expect these vectors to ultimately find applications for tight and reversible RNAi in mammalian cells in vivo.

Optimization and characterization of tRNA-shRNA expression constructs.

Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNALys3-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3′ tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.

An inducible system for expression and validation of the specificity of short hairpin RNA in mammalian cells

RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.

Spongiform Neurodegeneration-associated E3 Ligase Mahogunin Ubiquitylates TSG101 and Regulates Endosomal Trafficking

A null mutation in the gene encoding the putative E3 ubiquitin-protein ligase Mahogunin causes spongiform neurodegeneration, a recessively transmitted prion-like disease in mice. However, no substrates of Mahogunin have been identified, and the cellular role of Mahogunin is unknown. Here, we report the identification of TSG101, a key component of the endosomal sorting complex required for transport (ESCRT)-I, as a specific Mahogunin substrate. We find that Mahogunin interacts with the ubiquitin E2 variant (UEV) domain of TSG101 via its PSAP motif and that it catalyzes monoubiquitylation of TSG101 both in vivo and in vitro. Depletion of Mahogunin by small interfering RNAs in mammalian cells disrupts endosome-to-lysosome trafficking of epidermal growth factor receptor, resulting in prolonged activation of a downstream signaling cascade. Our findings support a role for Mahogunin in a proteasome-independent ubiquitylation pathway and suggest a link between dysregulation of endosomal trafficking and spongiform neurodegeneration.

Specific suppression of beta-secretase gene expression by short interfering RNA in mammalian cells

To investigate whether short interfering RNAs(siRNAs) of beta-site APP cleaving enzyme (BACE) can inhibit the expression of BACE in mammalian cells. The gene of EGFP, U6 promoter and beta-secretase targeting siRNA were cloned by PCR, respectively. The PCR products were inserted into plasmid pLXSN. The interfering vector pLXSN/EGFP-U6-siBACE was transferred into SK-N-SH cells to express BACE. The inhibition effect of BACE siRNA on BACE expression was investigated by fluoroscopy and immunohistochemistry method. The interfering vector pLXSN/EGFP-U6-siBACE was constructed successfully. The BACE siRNA inhibited the expression of BACE in the SK-N-SH cells specifically and effectively, and the production of A beta was reduced. BACE siRNA can inhibit the expression of BACE gene of mammalian cells.

Dominant-negative Pes1 mutants inhibit ribosomal RNA processing and cell proliferation via incorporation into the PeBoW-complex

The nucleolar PeBoW-complex, consisting of Pes1, Bop1 and WDR12, is essential for cell proliferation and processing of ribosomal RNA in mammalian cells. Here we have analysed the physical and functional interactions of Pes1 deletion mutants with the PeBoW-complex. Pes1 mutants M1 and M5, with N- and C-terminal truncations, respectively, displayed a dominant-negative phenotype. Both mutants showed nucleolar localization, blocked processing of the 36S/32S precursors to mature 28S rRNA, inhibited cell proliferation, and induced high p53 levels in proliferating, but not in resting cells. Mutant M1 and M5 proteins associated with large pre-ribosomal complexes and co-immunoprecipitated Bop1 and WDR12 proteins indicating their proper incorporation into the PeBoW-complex. We conclude that the dominant-negative effect of the M1 and M5 mutants is mediated by the impaired function of the PeBoW-complex.

Vector systems of RNA interference

RNA interference is a mechanism of posttranslational (at the level of mRNA) gene silencing. Sequence-specific mRNA degradation is realized with the help of small interfering RNAs produced by processing of a precursor using Dicer, an enzyme from the RNAse III family. This mechanism is now widely used in vitro on cultures of mammalian cells in order to elucidate functions of individual genes by gene specific knockdown. Analogs of small interference RNAs are intensely expressed during embryogenesis. The mechanism of RNA interference plays an especially important role in embryogenesis of invertebrates. Identification of the functions of small noncoding RNAs is essential for understanding the genetic mechanisms underlying individual developmental stages. In order to integrate small interference RNAs in mammalian cells, various systems have been developed that allow both transient (for 48 h) and stable expression in vitro. These systems are considered in the present review.