A genome-wide function of THSC/TREX-2 at active genes prevents transcription–replication collisions

The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3 end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription– replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis.Spanish Ministry of Economy and Competitiveness [BFU2010-16372]; Junta de Andalucía [CVI4567 and P12/BIO-1238]; European Union (FEDER); and a JAE predoctoral training grant from the Spanish Research Council (CSIC) [to J.M.S-P.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2010-16372].Peer reviewe

Cre-dependent DNA recombination activates a STING-dependent innate immune response

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies

Cre-dependent DNA recombination activates a STING-dependent innate immune response

Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies

Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors

Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2\u27-O-Methyl (2\u27OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2\u27OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2\u27OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application

Analysis of microRNA turnover in mammalian cells following Dicer1 ablation

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function

An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs

TLR7 gain-of-function genetic variation causes human lupus

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA and binds to guanosine. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP1 and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition

Modified Polyadenylation-Based RT-qPCR Increases Selectivity of Amplification of 3′-MicroRNA Isoforms

MicroRNA (miRNA) detection by reverse transcription (RT) quantitative real-time PCR (RT-qPCR) is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3′-end length variants (“isomiRs”), little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3′-end miRNA isoforms can exhibit 3′-length specific regulatory functions, underlining the need to develop strategies to differentiate 3′-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20–21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3′-isomiRs