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NAR Top Articles - Molecular Biology

Molecular Biology

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February 2015

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system
Bikard, D; Jiang, WY; Samai, P; Hochschild, A; Zhang, F; Marraffini, LA
Nucleic Acids Res. 2013, 41, 7429-7437
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The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
Ducani, C; Bernardinelli, G; Hogberg, B
Nucleic Acids Res. 2014, 42, 10596-10604
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In rolling circle replication, a circular template of DNA is replicated as a long single-stranded DNA concatamer that spools off when a strand displacing polymerase traverses the circular template. The current view is that this type of replication can only produce single-stranded DNA, because the only 3'-ends available are the ones being replicated along the circular templates. In contrast to this view, we find that rolling circle replication in vitro generates large amounts of double stranded DNA and that the production of single-stranded DNA terminates after some time. These properties can be suppressed by adding single-stranded DNA-binding proteins to the reaction. We conclude that amodel in which the polymerase switches templates to the already produced single-stranded DNA, with an exponential distribution of template switching, can explain the observed data. From this, we also provide an estimate value of the switching rate constant.

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival
Naro, C; Barbagallo, F; Chieffi, P; Bourgeois, CF; Paronetto, MP; Sette, C
Nucleic Acids Res. 2014, 42, 3218-3227
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NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase...

Landscape of target:guide homology effects on Cas9-mediated cleavage
Fu, BXH; Hansen, LL; Artiles, KL; Nonet, ML; Fire, AZ
Nucleic Acids Res. 2014, 42, 13778-13787
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To study target sequence specificity, selectivity, and reaction kinetics of Streptococcus pyogenes Cas9 activity, we challenged libraries of random variant targets with purified Cas9:: guide RNA complexes in vitro. Cleavage kinetics were nonlinear, with a burst of initial activity followed by slower sustained cleavage. Consistent with other recent analyses of Cas9 sequence specificity, we observe considerable (albeit incomplete) impairment of cleavage for targets mutated in the PAM sequence or in 'seed' sequences matching the proximal 8 bp of the guide. A second target region requiring close homology was located at the other end of the guide:: target duplex (positions 13-18 relative to the PAM). Sequences flanking the guide+PAM region had measurable (albeit modest) effects on cleavage. In addition, the first-base Guanine constraint commonly imposed by gRNA expression systems has little effect on overall cleavage efficiency. Taken together, these studies provide an in vitro understanding of the complexities of Cas9-gRNA interaction and cleavage beyond the general paradigm of site determination based on the 'seed' sequence and PAM.

Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system
Arslan, Z; Hermanns, V; Wurm, R; Wagner, R; Pul, U
Nucleic Acids Res. 2014, 42, 7884-7893
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The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5 '-ends of the repeat strands with the 3 '-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.

The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells
Gao, Y; Wu, FJ; Zhou, JC; Yan, L; Jurczak, MJ; Lee, HY; Yang, LH; Mueller, M; Zhou, XB; Dandolo, L; Szendroedi, J; Roden, M; Flannery, C; Taylor, H; Carmichael, GG; Shulman, GI; Huang, YQ
Nucleic Acids Res. 2014, 42, 13799-13811
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The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia down-regulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.

Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis
Koster, T; Meyer, K; Weinholdt, C; Smith, LM; Lummer, M; Speth, C; Grosse, I; Weigel, D; Staiger, D
Nucleic Acids Res. 2014, 42, 9925-9936
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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 14miRNAs with a minimum log(2) 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 vivo. Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels...

MiR-138 downregulates miRNA processing in HeLa cells by targeting RMND5A and decreasing Exportin-5 stability
Li, J; Chen, Y; Qin, XL; Wen, JZ; Ding, HM; Xia, W; Li, SH; Su, XT; Wang, W; Li, H; Zhao, Q; Fang, T; Qu, LH; Shao, NS
Nucleic Acids Res. 2014, 42, 458-474
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MicroRNAs (miRNAs) are a class of non-coding small RNAs that consist of similar to 22 nt and are involved in several biological processes by regulating target gene expression. MiR-138 has many biological functions and is often downregulated in cancers. Our results showed that overexpression of miR-138 downregulated target RMND5A (required for meiotic nuclear division 5 homolog A) and reduced Exportin-5 stability, which results in decreased levels of pre-miRNA nuclear export in HeLa cells. We also found that miR-138 could significantly inhibit HeLa cell migration by targeting RMND5A. Our study therefore identifies miR-138-RMND5A-Exportin-5 as a previously unknown miRNA processing regulatory pathway in HeLa cells.

IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)
Zirkel, A; Lederer, M; Stohr, N; Pazaitis, N; Huttelmaier, S
Nucleic Acids Res. 2013, 41, 6618-6636
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The oncofetal IGF2 mRNA-binding protein 1 (IGF2BP1) controls the migration and invasiveness of primary as well as tumor-derived cells in vitro. Whether the protein also modulates epithelial-mesenchymal-transition (EMT), a hallmark of tumor progression involved in tumor cell dissemination, remained elusive. In this study, we reveal that IGF2BP1 enhances mesenchymal-like cell properties in tumor-derived cells by promoting the expression of the transcriptional regulators LEF1 and SLUG (SNAI2). IGF2BP1 associates with LEF1 transcripts and prevents their degradation in a 3'-UTR-dependent manner resulting in an upregulation of LEF1 expression. LEF1 promotes transcription of the mesenchymal marker fibronectin by associating with the fibronectin 1 promoter. Moreover, LEF1 enforces the synthesis of the 'EMT-driving' transcriptional regulator SNAI2. Accordingly, IGF2BP1 knockdown causes MET-like (mesenchymal-epithelial-transition) morphological changes, enhances the formation of cell-cell contacts and reduces cell migration in various mesenchymal-like tumor-derived cells...

Activity-associated miRNA are packaged in Map1b-enriched exosomes released from depolarized neurons
Goldie, BJ; Dun, MD; Lin, MJ; Smith, ND; Verrills, NM; Dayas, CV; Cairns, MJ
Nucleic Acids Res. 2014, 42, 9195-9208
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Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity requiring complex mechanisms of post-transcriptional mRNA trafficking and regulation. Small non-coding miRNA are uniquely poised to support these functions by providing a nucleic-acid-based specificity component for universal-sequence-dependent RNA binding complexes. We investigated the subcellular distribution of these molecules in resting and potassium chloride depolarized human neuroblasts, and found both selective enrichment and depletion in neurites. Depolarization was associated with a neurite-restricted decrease in miRNA expression; a subset of these molecules was recovered from the depolarization medium in nuclease resistant extracellular exosomes. These vesicles were enriched with primate specific miRNA and the synaptic-plasticity-associated protein MAP1b. These findings further support a role for miRNA as neural plasticity regulators, as they are compartmentalized in neurons and undergo activity-associated redistribution or release into the extracellular matrix.

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