Gene Expression Profile Changes After Short-activating RNA-mediated Induction of Endogenous Pluripotency Factors in Human Mesenchymal Stem Cells

It is now recognized that small noncoding RNA sequences have the ability to mediate transcriptional activation of specific target genes in human cells. Using bioinformatics analysis and functional screening, we screened short-activating RNA (saRNA) oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors, Kruppel-like factor 4 (KLF4) and c-MYC. We identified KLF4 and c-MYC promoter-targeted saRNA sequences that consistently induced increases in their respective levels of nascent mRNA and protein expression in a time- and dose-dependent manner, as compared with scrambled sequence control oligonucleotides. The functional consequences of saRNA-induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis, which revealed significant increases in mRNA levels of their respective downstream pathway genes. Notably, the microarray profile after saRNA-mediated induction of endogenous KLF4 and c-MYC showed similar gene expression patterns for stem cell- and cell cycle-related genes as compared with lentiviral vector-mediated overexpression of exogenous KLF4 and c-MYC transgenes, while divergent gene expression patterns common to viral vector-mediated transgene delivery were also noted. The use of promoter-targeted saRNAs for the activation of pluripotency reprogramming factors could have broad implications for stem cell research

Distance constraints between microRNA target sites dictate efficacy and cooperativity

MicroRNAs (miRNAs) have the potential to regulate the expression of thousands of genes, but the mechanisms that determine whether a gene is targeted or not are poorly understood. We studied the genomic distribution of distances between pairs of identical miRNA seeds and found a propensity for moderate distances greater than about 13 nt between seed starts. Experimental data show that optimal down-regulation is obtained when two seed sites are separated by between 13 and 35 nt. By analyzing the distance between seed sites of endogenous miRNAs and transfected small interfering RNAs (siRNAs), we also find that cooperative targeting of sites with a separation in the optimal range can explain some of the siRNA off-target effects that have been reported in the literature

A role for human Dicer in pre-RISC loading of siRNAs

RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3′ overhangs and the thermodynamic properties of 2–4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA

Interplay between HIV-1 infection and host microRNAs

Using microRNA array analyses of in vitro HIV-1-infected CD4+ cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4+CD8− PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3′-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3′-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223

The Effect of Dicer Knockout on RNA Interference Using Various Dicer Substrate Small Interfering RNA (DsiRNA) Structures

Small interfering RNAs (siRNAs) are artificial molecules used to silence genes of interest through the RNA interference (RNAi) pathway, mediated by the endoribonuclease Dicer. Dicer-substrate small interfering RNAs (DsiRNAs) are an alternative to conventional 21-mer siRNAs, with an increased effectiveness of up to 100-fold compared to traditional 21-mer designs. DsiRNAs have a novel asymmetric design that allows them to be processed by Dicer into the desired conventional siRNAs. DsiRNAs are a useful tool for sequence-specific gene silencing, but the molecular mechanism underlying their increased efficacy is not precisely understood. In this study, to gain a deeper understanding of Dicer function in DsiRNAs, we designed nicked DsiRNAs with and without tetra-loops to target a specific mRNA sequence, established a Dicer knockout in the HCT116 cell line, and analyzed the efficacy of various DsiRNAs on RNAi-mediated gene silencing activity. The gene silencing activity of all DsiRNAs was reduced in Dicer knockout cells. We demonstrated that tetra-looped DsiRNAs exhibited increased efficacy for gene silencing, which was mediated by Dicer protein. Thus, this study improves our understanding of Dicer function, a key component of RNAi silencing, which will inform RNAi research and applications

Cell Cycle Abnormalities Associated with Differential Perturbations of the Human U5 snRNP Associated U5-200kD RNA Helicase

<div><p></p><p>Splicing of pre-messenger RNAs into functional messages requires a concerted assembly of proteins and small RNAs that identify the splice junctions and facilitate cleavage of exon-intron boundaries and ligation of exons. One of the key steps in the splicing reaction is the recruitment of a tri-snRNP harboring the U5/U4/U6 snRNPs. The U5 snRNP is also required for both steps of splicing and exon-exon joining. One of the key components of the tri-snRNP is the U5 200kd helicase. The human U5-200kD gene isolated from Hela cells encodes a 200 kDa protein with putative RNA helicase function. Surprisingly, little is known about the functional role of this protein in humans. Therefore, we have investigated the role of the U5-200kD RNA helicase in mammalian cell culture. We created and expressed a dominant negative domain I mutant of the RNA helicase in HEK293 cells and used RNAi to downregulate expression of the endogenous protein. Transient and stable expression of the domain I mutant U5-200kD protein using an ecdysone-inducible system and transient expression of an anti-U5-200kD short hairpin RNA (shRNA) resulted in differential splicing and growth defects in the 293/EcR cells. Cell cycle analysis of the dominant negative clones revealed delayed exit from the G2/M phase of the cell cycle due to a mild splicing defect. In contrast to the domain I dominant negative mutant expressing cells, transient expression of an anti-U5-200kD shRNA resulted in a pronounced S phase arrest and a minute splicing defect. Collectively, this work demonstrates for the first time establishment of differential human cell culture splicing and cell cycle defect models due to perturbed levels of an essential core splicing factor.</p></div

Stable expression of the mutant U5-200kD protein results in growth defects.

<p>A) <i>Growth defects in the Ponasterone A inducible, stably transfected mutant U5-200kD clones.</i> The uninduced and induced 293/EcR cells show a normal, wild type growth rate, indicating that the Ponasterone A had no effect on growth rate. p<0.001. B) <i>Detection of the mutant U5-200kD gene in SMC10 cells using mutant specific primers.</i> The mutant specific primers detected the mutant U5-200kD mRNA only in the SMC10 uninduced (lane 7 lower band) and induced (lane 9 lower band) cells. The top bands in lanes 3, 5, 7 and 9 are the histone PCR products. Lanes 2, 4, 6 and 8 are minus RT controls showing lack of DNA contamination. Lanes 1 and 10 are the 100 bp markers.</p

SMC10 cells have altered growth patterns and abnormal cell morphologies.

<p>A) <i>Growth pattern and size differences between the parental 293/EcR and stable mutant clone 10 cell lines.</i> B) <i>Comparisons of the growth morphologies of the 293/EcR and SMC10 cells.</i> a) 293/EcR cells undergoing cell division. b) 293/EcR cells prior to cytokinesis. c and d) SMC10 cells remain attached in dumbbell like structures. C) <i>Cell cycle analyses of the uninduced and Ponasterone A treated C13 and SMC10 cell lines.</i> In the SMC10 cells the number of cells in the G2/M phase has increased compared to the control 293/EcR cell lines. p<0.05. D) <i>Comparison of the G2/M and sub G1 phases of C13 and SMC10 cell lines.</i> The SMC10 cells are stalled at the G2/M phase. p<0.05.</p

siRNA and shRNA knockdown of the U5-200kD mRNA.

<p>A) <i>Diagram of the site II siRNA and site V siRNA and shRNA targeting the N-terminal coding region of the U5-200kD mRNA.</i> B) <i>RT-PCR analyses of the 293/EcR cells transfected with anti-U5-200kD site II and V siRNAs and the irrelevant anti HIV-1 Rev siRNA.</i> The lower band in lanes 2, 4 and 6 is the RT-PCR amplified endogenous U5-200kD mRNA. The upper band is the RT-PCR amplified endogenous H2A which was used to normalize the expression of the U5-200kD mRNA. Lanes 1, 3 and 5 represent the RT-PCR reactions without addition of reverse-transcriptase. C) <i>Semi-quantitative analysis of the U5-200kD RT-PCR reactions.</i> After normalizing the U5-200kD signal to the histone signal, the bar graph for both site V and II treated cells indicate approximately forty-five percent reduction of the U5-200kD mRNA in the total isolated cellular RNA. Considering that the transfection efficiency for this experiment was approximately fifty percent, we assume close to one hundred percent reduction of the target mRNA in the transfected cells. Both sites V and II siRNAs were effective in reducing the U5-200kD mRNA in the 293/EcR cells.</p

Expression analysis of the ecdysone-inducible mutant U5-200kD gene in HEK293 cells.

<p>A) <i>Organization of the regulatory elements in the ecdysone-inducible plasmid as well as the GKT-DNT inducible mutant U5-200kD, pI-M11.</i> Numbers 1-2136 indicate the amino acids in the ORF. PA is the polyadenylation site and ΔHSP is the minimal heat shock promoter on the ecdysone-inducible plasmid. B) <i>Transient expression of the inducible mutant U5-200kD gene.</i> The mutant U5-200kD gene (M11) cloned under the control of the ecdysone-inducible plasmid (pI-M11) was transfected into the trans-activator 293/EcR cell line clone (C13). The mutant gene was induced with 0.5, 1.0 and 3.0 mM of the inducer, Ponasterone A. For the controls, the enhanced green florescent protein (EGFP) was cloned under the inducible promoter and transfected into the C13 cell line (pIE-3.0 mM), 3.0 mM Ponasterone A was added to the C13 cell line (C13–3.0 mM) and the pIM11 plasmid was transfected into the C13 cell line without the inducer (pIM11-0 mM). The cell count analyses revealed growth retardation in the induced M11 dishes and not in the control plates. C) Western analysis of mutant U5-200KD protein expression. Histone 2A (H2A) was included as a loading control. D) <i>RT-PCR detection of the transiently transfected mutant U5-200kD gene.</i> Lanes 1 and 6 are the 100 bp ladders. Lane 2 is the RT-PCR reaction of the uninduced pI-M11 transfected into the 293/EcR cells. Lane 4 is the RT-PCR reaction of the induced pI-M11 transfected into the 293/EcR cells. Lanes 3 and 5 are the minus RT reactions for lanes 2 and 4, respectively.</p