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Source: Nucleic Acids Research
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Total 88 results found since Jan 2013.
Visualization of self-delivering hydrophobically modified siRNA cellular internalization
siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation ...
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Ly, S., Navaroli, D. M., Didiot, M.-C., Cardia, J., Pandarinathan, L., Alterman, J. F., Fogarty, K., Standley, C., Lifshitz, L. M., Bellve, K. D., Prot, M., Echeverria, D., Corvera, S., Khvorova, A. Tags: Chemical Biology and Nucleic Acid Chemistry Source Type: research
RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns
We present here a comprehensive RNA-seq study that covers multiple tissues in an SMA mouse model. We show elevated U12-intron retention in all examined tissues from SMA mice, and that U12-dependent intron retention is induced upon siRNA knock-down of SMN in HeLa cells. Furthermore, we show that retention of U12-dependent introns is mitigated by ASO treatment of SMA mice and that many transcriptional changes are reversed. Finally, we report on missplicing of several Ca2+ channel genes that may explain disrupted Ca2+ homeostasis in SMA and activation of Cdk5.
Source: Nucleic Acids Research - January 8, 2017 Category: Research Authors: Doktor, T. K., Hua, Y., Andersen, H. S., Broner, S., Liu, Y. H., Wieckowska, A., Dembic, M., Bruun, G. H., Krainer, A. R., Andresen, B. S. Tags: RNA Source Type: research
Structural basis for single-stranded RNA recognition and cleavage by C3PO
Translin and translin-associated factor-x are highly conserved in eukaroytes; they can form heteromeric complexes (known as C3POs) and participate in various nucleic acid metabolism pathways. In humans and Drosophila, C3POs cleave the fragmented siRNA passenger strands and facilitate the activation of RNA-induced silencing complex, the effector complex of RNA interference (RNAi). Here, we report three crystal structures of Nanoarchaeum equitans (Ne) C3PO. The apo-NeC3PO structure adopts an open form and unravels a potential substrates entryway for the first time. The NeC3PO:ssRNA and NeC3PO:ssDNA complexes fold like closed...
Source: Nucleic Acids Research - October 29, 2016 Category: Research Authors: Zhang, J., Liu, H., Yao, Q., Yu, X., Chen, Y., Cui, R., Wu, B., Zheng, L., Zuo, J., Huang, Z., Ma, J., Gan, J. Tags: Structural Biology Source Type: research
Homology directed repair is unaffected by the absence of siRNAs in Drosophila melanogaster
Small interfering RNAs (siRNAs) defend the organism against harmful transcripts from exogenous (e.g. viral) or endogenous (e.g. transposons) sources. Recent publications describe the production of siRNAs induced by DNA double-strand breaks (DSB) in Neurospora crassa, Arabidopsis thaliana, Drosophila melanogaster and human cells, which suggests a conserved function. A current hypothesis is that break-induced small RNAs ensure efficient homologous recombination (HR). However, biogenesis of siRNAs is often intertwined with other small RNA species, such as microRNAs (miRNAs), which complicates interpretation of experimental re...
Source: Nucleic Acids Research - September 28, 2016 Category: Research Authors: Schmidts, I., Böttcher, R., Mirkovic-Hösle, M., Förstemann, K. Tags: Genome Integrity, Repair and Replication Source Type: research
Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs
Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3' modifications (truncations and non-templated tailing) of plant miRNAs when the 2'-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3' ends of both miRNAs and endo...
Source: Nucleic Acids Research - September 1, 2016 Category: Research Authors: Wang, F., Johnson, N. R., Coruh, C., Axtell, M. J. Tags: RNA Source Type: research
The delivery of therapeutic oligonucleotides
The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for e...
Source: Nucleic Acids Research - August 17, 2016 Category: Research Authors: Juliano, R. L. Tags: SURVEY AND SUMMARY Source Type: research
Comprehensive analysis of high-throughput screens with HiTSeekR
High-throughput screening (HTS) is an indispensable tool for drug (target) discovery that currently lacks user-friendly software tools for the robust identification of putative hits from HTS experiments and for the interpretation of these findings in the context of systems biology. We developed HiTSeekR as a one-stop solution for chemical compound screens, siRNA knock-down and CRISPR/Cas9 knock-out screens, as well as microRNA inhibitor and -mimics screens. We chose three use cases that demonstrate the potential of HiTSeekR to fully exploit HTS screening data in quite heterogeneous contexts to generate novel hypotheses for...
Source: Nucleic Acids Research - August 17, 2016 Category: Research Authors: List, M., Schmidt, S., Christiansen, H., Rehmsmeier, M., Tan, Q., Mollenhauer, J., Baumbach, J. Tags: Computational Methods, Genomics Data Resources and Analyses Source Type: research
Two sets of RNAi components are required for heterochromatin formation in trans triggered by truncated transgenes
Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci in trans in the ciliate Paramecium. We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this...
Source: Nucleic Acids Research - July 6, 2016 Category: Research Authors: Götz, U., Marker, S., Cheaib, M., Andresen, K., Shrestha, S., Durai, D. A., Nordström, K. J., Schulz, M. H., Simon, M. Tags: RNA Source Type: research
Novel glycylated sugar alcohols protect ESC-specific microRNAs from degradation in iPS cells
Excessive accumulation of embryonic stem cell (ESC)-specific microRNAs occurs in both ESCs and induced pluripotent stem cells (iPSC); yet, the mechanism involved is unknown. In iPSCs, we for the first time found that novel glycylated sugar alcohols, particularly glycylglycerins, are tightly bound with ESC-specific microRNA precursors (pre-miRNA), such as pre-miR-302. Among these isolated glycylglycerins, we further identified that 1,3-diglycylglycerin and 1,2,3-triglycylglycerin are two major compounds bonded with negatively charged nucleic acids via electro-affinity and subsequently forming sugar-like coats in the hairpin...
Source: Nucleic Acids Research - May 31, 2016 Category: Research Authors: Chang-Lin, S., Hung, A., Chang, D. C., Lin, Y.-W., Ying, S.-Y., Lin, S.-L. Tags: RNA Source Type: research
Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus
Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruite...
Source: Nucleic Acids Research - April 19, 2016 Category: Research Authors: Yang, R., Kerschner, J. L., Gosalia, N., Neems, D., Gorsic, L. K., Safi, A., Crawford, G. E., Kosak, S. T., Leir, S.-H., Harris, A. Tags: Gene regulation, Chromatin and Epigenetics Source Type: research
RNA cleavage products generated by antisense oligonucleotides and siRNAs are processed by the RNA surveillance machinery
DNA-based antisense oligonucleotides (ASOs) elicit cleavage of the targeted RNA by the endoribonuclease RNase H1, whereas siRNAs mediate cleavage through the RNAi pathway. To determine the fates of the cleaved RNA in cells, we lowered the levels of the factors involved in RNA surveillance prior to treating cells with ASOs or siRNA and analyzed cleavage products by RACE. The cytoplasmic 5' to 3' exoribonuclease XRN1 was responsible for the degradation of the downstream cleavage products generated by ASOs or siRNA targeting mRNAs. In contrast, downstream cleavage products generated by ASOs targeting nuclear long non-coding R...
Source: Nucleic Acids Research - April 19, 2016 Category: Research Authors: Lima, W. F., De Hoyos, C. L., Liang, X.-h., Crooke, S. T. Tags: RNA Source Type: research
Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms
DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assa...
Source: Nucleic Acids Research - April 6, 2016 Category: Research Authors: Kostyrko, K., Mermod, N. Tags: Recombination, Repair Methods Online Source Type: research
LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner
Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation o...
Source: Nucleic Acids Research - January 29, 2016 Category: Research Authors: Kramer, H. B., Lai, C.-F., Patel, H., Periyasamy, M., Lin, M.-L., Feller, S. M., Fuller-Pace, F. V., Meek, D. W., Ali, S., Buluwela, L. Tags: Gene regulation, Chromatin and Epigenetics Source Type: research
Cold-inducible RNA-binding protein CIRP/hnRNP A18 regulates telomerase activity in a temperature-dependent manner
The telomerase is responsible for adding telomeric repeats to chromosomal ends and consists of the reverse transcriptase TERT and the RNA subunit TERC. The expression and activity of the telomerase are tightly regulated, and aberrant activation of the telomerase has been observed in >85% of human cancers. To better understand telomerase regulation, we performed immunoprecipitations coupled with mass spectrometry (IP-MS) and identified cold inducible RNA-binding protein (CIRP or hnRNP A18) as a telomerase-interacting factor. We have found that CIRP is necessary to maintain telomerase activities at both 32°C and 37&de...
Source: Nucleic Acids Research - January 29, 2016 Category: Research Authors: Zhang, Y., Wu, Y., Mao, P., Li, F., Han, X., Zhang, Y., Jiang, S., Chen, Y., Huang, J., Liu, D., Zhao, Y., Ma, W., Songyang, Z. Tags: Molecular Biology Source Type: research
RNAi revised - target mRNA-dependent enhancement of gene silencing
The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy.
We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence...
Source: Nucleic Acids Research - December 15, 2015 Category: Research Authors: Dornseifer, S., Willkomm, S., Far, R. K.-K., Liebschwager, J., Beltsiou, F., Frank, K., Laufer, S. D., Martinetz, T., Sczakiel, G., Claussen, J. C., Restle, T. Tags: Computational Biology Source Type: research
