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NAR Top Articles - Molecular Biology

Molecular Biology

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September 2015

Dual role of transcription and transcript stability in the regulation of gene expression in Escherichia coli cells cultured on glucose at different growth rates
Esquerre, T; Laguerre, S; Turlan, C; Carpousis, AJ; Girbal, L; Cocaign-Bousquet, M
Nucleic Acids Res. 2014, 42, 2460-2472
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Microorganisms extensively reorganize gene expression to adjust growth rate to changes in growth conditions. At the genomic scale, we measured the contribution of both transcription and transcript stability to regulating messenger RNA (mRNA) concentration in Escherichia coli. Transcriptional control was the dominant regulatory process. Between growth rates of 0.10 and 0.63 h(-1), there was a generic increase in the bulk mRNA transcription. However, many transcripts became less stable and the median mRNA half-life decreased from 4.2 to 2.8 min. This is the first evidence that mRNA turnover is slower at extremely low-growth rates. The destabilization of many, but not all, transcripts at high-growth rate correlated with transcriptional upregulation of genes encoding the mRNA degradation machinery. We identified five classes of growth-rate regulation ranging from mainly transcriptional to mainly degradational. In general, differential stability within polycistronic messages encoded by operons does not appear to be affected by growth rate...

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.

LHX2 regulates the neural differentiation of human embryonic stem cells via transcriptional modulation of PAX6 and CER1
Hou, PS; Chuang, CY; Kao, CF; Chou, SJ; Stone, L; Ho, HN; Chien, CL; Kuo, HC
Nucleic Acids Res. 2013, 41, 7753-7770
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The LIM homeobox 2 transcription factor Lhx2 is known to control crucial aspects of neural development in various species. However, its function in human neural development is still elusive. Here, we demonstrate that LHX2 plays a critical role in human neural differentiation, using human embryonic stem cells (hESCs) as a model. In hESC-derived neural progenitors (hESC-NPs), LHX2 was found to be expressed before PAX6, and co-expressed with early neural markers. Conditional ectopic expression of LHX2 promoted neural differentiation, whereas disruption of LHX2 expression in hESCs significantly impaired neural differentiation. Furthermore, we have demonstrated that LHX2 regulates neural differentiation at two levels: first, it promotes expression of PAX6 by binding to its active enhancers, and second, it attenuates BMP and WNT signaling by promoting expression of the BMP and WNT antagonist Cerberus 1 gene (CER1), to inhibit non-neural differentiation. These findings indicate that LHX2 regulates the transcription of downstream intrinsic and extrinsic molecules that are essential for early neural differentiation in human.

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.

Differential regulation by ppGpp versus pppGpp in Escherichia coli
Mechold, U; Potrykus, K; Murphy, H; Murakami, KS; Cashel, M
Nucleic Acids Res. 2013, 41, 6175-6189
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Both ppGpp and pppGpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. There are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppGpp and pppGpp. Here, we achieve preferential accumulation of either ppGpp or pppGpp with Escherichia coli strains through induction of different Streptococcal (p)ppGpp synthetase fragments. In addition, expression of E. coli GppA, a pppGpp 5'-gamma phosphate hydrolase that converts pppGpp to ppGpp, is manipulated to fine tune differential accumulation of ppGpp and pppGpp. In vivo and in vitro experiments show that pppGpp is less potent than ppGpp with respect to regulation of growth rate, RNA/DNA ratios, ribosomal RNA P1 promoter transcription inhibition, threonine operon promoter activation and RpoS induction. To provide further insights into regulation by (p)ppGpp, we have also determined crystal structures of E. coli RNA polymerase-Sigma(70) holoenzyme with ppGpp and pppGpp...

The RIG-I ATPase core has evolved a functional requirement for allosteric stabilization by the Pincer domain
Rawling, DC; Kohlway, AS; Luo, DH; Ding, SC; Pyle, AM
Nucleic Acids Res. 2014, 42, 11601-11611
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Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor expressed in metazoan cells that is responsible for eliciting the production of type I interferons and pro-inflammatory cytokines upon detection of intracellular, non-self RNA. Structural studies of RIG-I have identified a novel Pincer domain composed of two alpha helices that physically tethers the C-terminal domain to the SF2 helicase core. We find that the Pincer plays an important role in mediating the enzymatic and signaling activities of RIG-I. We identify a series ofmutations that additively decouple the Pincer motif from the ATPase core and show that this decoupling results in impaired signaling. Through enzymological and biophysical analysis, we further show that the Pincer domain controls coupled enzymatic activity of the protein through allosteric control of the ATPase core. Further, we show that select regions of the HEL1 domain have evolved to potentiate interactions with the Pincer domain, resulting in an adapted ATPase cleft that is now responsive to adjacent domains that selectively bind viral RNA.

The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication
Atkinson, NJ; Witteveldt, J; Evans, DJ; Simmonds, P
Nucleic Acids Res. 2014, 42, 4527-4545
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Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-beta, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants...

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.

HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway
Xue, M; Yao, SH; Hu, MM; Li, W; Hao, TT; Zhou, F; Zhu, XF; Lu, HM; Qin, D; Yan, Q; Zhu, JZ; Gao, SJ; Lu, C
Nucleic Acids Res. 2014, 42, 9862-9879
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Kaposi's sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV). Although the interaction between HIV-1 and KSHV plays a pivotal role in promoting the aggressive manifestations of KS, the pathogenesis underlying AIDS-KS remains largely unknown. Here we examined HIV-1 Nef protein promotion of KSHV oncoprotein K1-induced angiogenesis. We showed that both internalized and ectopic expression of Nef in endothelial cells synergized with K1 to facilitate vascular tube formation and cell proliferation, and enhance angiogenesis in a chicken CAM model. In vivo experiments further indicated that Nef accelerated K1-induced angiogenesis and tumorigenesis in athymic nu/nu mice. Mechanistic studies revealed that Nef and K1 synergistically activated PI3K/AKT/mTOR signaling by downregulating PTEN. Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3' UTR of its mRNA. Inhibition of miR-718 expression increased PTEN synthesis...

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