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Source: Nucleic Acids Research
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Total 88 results found since Jan 2013.
Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis
The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ±0.5. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vi...
Source: Nucleic Acids Research - September 1, 2014 Category: Research Authors: Koster, T., Meyer, K., Weinholdt, C., Smith, L. M., Lummer, M., Speth, C., Grosse, I., Weigel, D., Staiger, D. Tags: Molecular Biology Source Type: research
Computational detection and suppression of sequence-specific off-target phenotypes from whole genome RNAi screens
A challenge for large-scale siRNA loss-of-function studies is the biological pleiotropy resulting from multiple modes of action of siRNA reagents. A major confounding feature of these reagents is the microRNA-like translational quelling resulting from short regions of oligonucleotide complementarity to many different messenger RNAs. We developed a computational approach, deconvolution analysis of RNAi screening data, for automated quantitation of off-target effects in RNAi screening data sets. Substantial reduction of off-target rates was experimentally validated in five distinct biological screens across different genome-...
Source: Nucleic Acids Research - July 31, 2014 Category: Research Authors: Zhong, R., Kim, J., Kim, H. S., Kim, M., Lum, L., Levine, B., Xiao, G., White, M. A., Xie, Y. Tags: Computational Methods, Targeted inhibition of gene function Computational Biology Source Type: research
Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi
RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthe...
Source: Nucleic Acids Research - July 31, 2014 Category: Research Authors: van Cleef, K. W. R., van Mierlo, J. T., Miesen, P., Overheul, G. J., Fros, J. J., Schuster, S., Marklewitz, M., Pijlman, G. P., Junglen, S., van Rij, R. P. Tags: Structural Biology Source Type: research
The protein kinase TOUSLED facilitates RNAi in Arabidopsis
RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are ju...
Source: Nucleic Acids Research - July 3, 2014 Category: Research Authors: Uddin, M. N., Dunoyer, P., Schott, G., Akhter, S., Shi, C., Lucas, W. J., Voinnet, O., Kim, J.-Y. Tags: RNA Source Type: research
siPools: highly complex but accurately defined siRNA pools eliminate off-target effects
Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools—referred to as siPools—containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by th...
Source: Nucleic Acids Research - July 3, 2014 Category: Research Authors: Hannus, M., Beitzinger, M., Engelmann, J. C., Weickert, M.-T., Spang, R., Hannus, S., Meister, G. Tags: RNA Source Type: research
Staufen1-mediated mRNA decay induces Requiem mRNA decay through binding of Staufen1 to the Requiem 3'UTR
Requiem (REQ/DPF2) was originally identified as an apoptosis-inducing protein in mouse myeloid cells and belongs to the novel Krüppel-type zinc finger d4-protein family of proteins, which includes neuro-d4 (DPF1) and cer-d4 (DPF3). Interestingly, when a portion of the REQ messenger ribonucleic acid (mRNA) 3' untranslated region (3'UTR), referred to as G8, was overexpressed in K562 cells, β-globin expression was induced, suggesting that the 3'UTR of REQ mRNA plays a physiological role. Here, we present evidence that the REQ mRNA 3'UTR, along with its trans-acting factor, Staufen1 (STAU1), is able to reduce the lev...
Source: Nucleic Acids Research - June 19, 2014 Category: Research Authors: Kim, M. Y., Park, J., Lee, J. J., Ha, D. H., Kim, J., Kim, C. G., Hwang, J., Kim, C. G. Tags: Gene regulation, Chromatin and Epigenetics Source Type: research
A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3' untranslated region-mediated gene regulation
Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3' untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3' UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regula...
Source: Nucleic Acids Research - June 2, 2014 Category: Research Authors: Chaudhury, A., Kongchan, N., Gengler, J. P., Mohanty, V., Christiansen, A. E., Fachini, J. M., Martin, J. F., Neilson, J. R. Tags: Methods Online Source Type: research
Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs
RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3'-CH2-CO-NH-5' amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typi...
Source: Nucleic Acids Research - June 2, 2014 Category: Research Authors: Mutisya, D., Selvam, C., Lunstad, B. D., Pallan, P. S., Haas, A., Leake, D., Egli, M., Rozners, E. Tags: RNA Source Type: research
miR-Synth: a computational resource for the design of multi-site multi-target synthetic miRNAs
RNAi is a powerful tool for the regulation of gene expression. It is widely and successfully employed in functional studies and is now emerging as a promising therapeutic approach. Several RNAi-based clinical trials suggest encouraging results in the treatment of a variety of diseases, including cancer. Here we present miR-Synth, a computational resource for the design of synthetic microRNAs able to target multiple genes in multiple sites. The proposed strategy constitutes a valid alternative to the use of siRNA, allowing the employment of a fewer number of molecules for the inhibition of multiple targets. This may represe...
Source: Nucleic Acids Research - May 19, 2014 Category: Research Authors: Lagana, A., Acunzo, M., Romano, G., Pulvirenti, A., Veneziano, D., Cascione, L., Giugno, R., Gasparini, P., Shasha, D., Ferro, A., Croce, C. M. Tags: Computational Biology Source Type: research
Systematic design and functional analysis of artificial microRNAs
Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2–8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and fun...
Source: Nucleic Acids Research - May 19, 2014 Category: Research Authors: Arroyo, J. D., Gallichotte, E. N., Tewari, M. Tags: Synthetic Biology and Chemistry Source Type: research
Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins, Ago and TRBP, in mammalian cells
Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5' proximal nucleotides, which include seed sequence (positions 2–8 from the 5' end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which t...
Source: Nucleic Acids Research - April 30, 2014 Category: Research Authors: Takahashi, T., Zenno, S., Ishibashi, O., Takizawa, T., Saigo, K., Ui-Tei, K. Tags: RNA Source Type: research
RNase L restricts the mobility of engineered retrotransposons in cultured human cells
Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2',5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (N...
Source: Nucleic Acids Research - April 2, 2014 Category: Research Authors: Zhang, A., Dong, B., Doucet, A. J., Moldovan, J. B., Moran, J. V., Silverman, R. H. Tags: Nucleic Acid Enzymes Source Type: research
Dicer-related helicase 3 forms an obligate dimer for recognizing 22G-RNA
Dicer is a specialized nuclease that produces RNA molecules of specific lengths for use in gene silencing pathways. Dicer relies on the correct measurement of RNA target duplexes to generate products of specific lengths. It is thought that Dicer uses its multidomain architecture to calibrate RNA product length. However, this measurement model is derived from structural information from a protozoan Dicer, and does not account for the helicase domain present in higher organisms. The Caenorhabditis elegans Dicer-related helicase 3 (DRH-3) is an ortholog of the Dicer and RIG-I family of double-strand RNA activated ATPases esse...
Source: Nucleic Acids Research - April 2, 2014 Category: Research Authors: Fitzgerald, M. E., Vela, A., Pyle, A. M. Tags: Nucleic Acid Enzymes Source Type: research
Glycogen synthase kinase 3 beta inhibits microRNA-183-96-182 cluster via the {beta}-Catenin/TCF/LEF-1 pathway in gastric cancer cells
Glycogen synthase kinase 3 beta (GSK3β) is a critical protein kinase that phosphorylates numerous proteins in cells and thereby impacts multiple pathways including the β-Catenin/TCF/LEF-1 pathway. MicroRNAs (miRs) are a class of noncoding small RNAs of ~22 nucleotides in length. Both GSK3β and miR play myriad roles in cell functions including stem cell development, apoptosis, embryogenesis and tumorigenesis. Here we show that GSK3β inhibits the expression of miR-96, miR-182 and miR-183 through the β-Catenin/TCF/LEF-1 pathway. Knockout of GSK3β in mouse embryonic fibroblast cells increases expr...
Source: Nucleic Acids Research - March 11, 2014 Category: Research Authors: Tang, X., Zheng, D., Hu, P., Zeng, Z., Li, M., Tucker, L., Monahan, R., Resnick, M. B., Liu, M., Ramratnam, B. Tags: Gene Regulation, Chromatin and Epigenetics Source Type: research
Distinct substrate specificities of Arabidopsis DCL3 and DCL4
In Arabidopsis thaliana, Dicer-like 3 (DCL3) and Dicer-like 4 (DCL4) cleave long, perfect double-stranded RNAs (dsRNAs) into 24 and 21 nucleotides (nt) small interfering RNAs, respectively, which in turn function in RNA-directed DNA methylation and RNA interference, respectively. To reveal how DCL3 and DCL4 individually recognize long perfect dsRNAs as substrates, we biochemically characterized DCL3 and DCL4 and compared their enzymatic properties. DCL3 preferentially cleaves short dsRNAs with 5' phosphorylated adenosine or uridine and a 1 nt 3' overhang, whereas DCL4 cleaves long dsRNAs with blunt ends or with a 1 or 2 nt...
Source: Nucleic Acids Research - February 10, 2014 Category: Research Authors: Nagano, H., Fukudome, A., Hiraguri, A., Moriyama, H., Fukuhara, T. Tags: Nucleic Acid Enzymes Source Type: research
