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Post-translational modification by the small ubiquitin-like modifier (SUMO) regulates the cellular response to different types of stress and plays a pivotal role in the control of oncogenic viral infections. Here we investigated the capacity of microRNAs (miRNAs) encoded by Epstein-Barr virus to interfere with the SUMO signaling network. Using a computational strategy that scores different properties of miRNA-mRNA target pairs, we identified a minimal set of 575 members of the SUMO interactome that may be targeted by one or more Epstein-Barr virus miRNAs. A significant proportion of the candidates cluster in a functional network that controls chromatin organization, stress, DNA damage and immune responses, apoptosis and transforming growth factor beta signaling. Multiple components of the transforming growth factor beta signaling pathway were inhibited upon upregulation of the BamHI-H rightward open reading frame 1 (BHRF1) encoded miRNAs in cells transduced with recombinant lentiviruses or entering the productive virus cycle. These findings point to the capacity of viral miRNAs to interfere with SUMO-regulated cellular functions that control key aspects of viral replication and pathogenesis.

Antisense agents that target growth-essential genes display surprisingly potent bactericidal properties. In particular, peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers linked to cationic carrier peptides are effective in time kill assays and as inhibitors of bacterial peritonitis in mice. It is unclear how these relatively large antimicrobials overcome stringent bacterial barriers and mediate killing. Here we determined the transit kinetics of peptide–PNAs and observed an accumulation of cell-associated PNA in Escherichia coli and slow efflux. An inhibitor of drug efflux pumps did not alter peptide–PNA potency, indicating a lack of active efflux from cells. Consistent with cell retention, the post-antibiotic effect (PAE) of the anti-acyl carrier protein (acpP) peptide–PNA was greater than 11 hours. Bacterial cell accumulation and a long PAE are properties of significant interest for antimicrobial development.

A combination of single-cell transcriptomics with in situ hybridization information enables single cells to be positioned within their tissue.

Single-cell gene expression analyses hold promise for characterizing cellular heterogeneity, but current methods compromise on either the coverage, the sensitivity or the throughput. Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. Smart-seq2 transcriptome libraries have improved detection, coverage, bias and accuracy compared to Smart-seq libraries and are generated with off-the-shelf reagents at lower cost.

Genome-wide transcriptome analyses are routinely used to monitor tissue-, disease- and cell type–specific gene expression, but it has been technically challenging to generate expression profiles from single cells. Here we describe a robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which enhances detailed analyses of alternative transcript isoforms and identification of single-nucleotide polymorphisms. We determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series. We found that although gene expression estimates from single cells have increased noise, hundreds of differentially expressed genes could be identified using few cells per cell type. Applying Smart-Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including candidate biomarkers for melanoma circulating tumor cells. Our protocol will be useful for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells.

The large tegument proteins of herpesviruses encode conserved cysteine proteases of unknown function. Here we show that BPLF1, the Epstein-Barr-virus-encoded member of this protease family, is a deneddylase that regulates virus production by modulating the activity of cullin-RING ligases (CRLs). BPLF1 hydrolyses NEDD8 conjugates in vitro, acts as a deneddylase in vivo, binds to cullins and stabilizes CRL substrates. Expression of BPLF1 alone or in the context of the productive virus cycle induces accumulation of the licensing factor CDT1 and deregulates S-phase DNA synthesis. Inhibition of BPLF1 during the productive virus cycle prevents cellular DNA re-replication and inhibits virus replication. Viral DNA synthesis is restored by overexpression of CDT1. Homologues encoded by other herpesviruses share the deneddylase activity. Thus, these enzymes are likely to have a key function in the virus life cycle by inducing a replication-permissive S-phase-like cellular environment.

Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription–polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells.

Short regulatory RNAs are widespread in bacteria,and many function through antisense recognitionof mRNA. Among the best studied antisensetranscripts are RNA antitoxins that repress toxinmRNA translation. The hok/sok locus of plasmid R1from Escherichia coli is an established model forRNA antitoxin action. Base-pairing between hokmRNA and Sok-antisense-RNA increases plasmidmaintenance through post-segregational-killing ofplasmid-free progeny cells. To test the model andthe idea that sequestration of Sok-RNA activity couldprovide a novel antimicrobial strategy, we designedanti Sok peptide nucleic acid (PNA) oligomers that,according to the model, would act as competitiveinhibitors of hok mRNA::Sok-RNA interactions. Inhok/sok-carrying cells, anti Sok PNAs were morebactericidal than rifampicin. Also, anti Sok PNAsinduced ghost cell morphology and an accumulationof mature hok mRNA, consistent with cell killingthrough synthesis of Hok protein. The results sup-port the sense/antisense model for hok mRNArepression by Sok-RNA and demonstrate that anti-sense agents can be used to out-compete RNA::RNAinteractions in bacteria. Finally, BLAST analyses of 200 prokaryotic genomes revealed that manyenteric bacteria have multiple hok/sok homologousand analogous RNA-regulated toxin–antitoxin loci.Therefore, it is possible to activate suicide in bacteriaby targeting antitoxins.INTRODUCTIONNon-coding regulatory RNAs are widely expressed in manygenomes (1,2). A large number of non-coding RNAs arecomplementary to active open reading frames, yet there isonly limited evidence for direct sense/antisense interactions.Antisense transcripts are encoded both in-cis and in-transand are believed to modulate RNA processing, decay andtranslation through direct pairing with complementary targetsequences (3). Bacterial genomes and plasmids contain anumber of annotated as well as predicted sense and antisensegenes. Despite predictions of widespread sense/antisensepairing in several species (4–7), there have been few attemptsto experimentally probe these structures and test the effects ofdisrupted interactions (8).A paradigm for sense/antisense RNA pairing is the hok/soktoxin–antitoxin (TA) plasmid stabilization locus of theR1 plasmid in E. coli (9). The hok/sok locus codes for threegenes: hok (host killing) encodes a highly toxic trans-membrane protein that irreversibly damages the cell mem-brane (10). The mok (modulation of killing) reading frameoverlaps with hok sequences and is required for hok expres-sion and translation. Finally, the sok (suppression of killing)gene encodes a small antisense RNA in-cis that blocks trans-lation of the mok reading frame and thus inhibits expressionof hokmRNA (11). Pairing between Sok and transcriptsis supported by in vitro and phylogenetic studies (12–14).Sok-RNA is very unstable (half-life in the order of 30 s)but driven by a strong promoter. In contrast, the full-lengthhok transcript is heavily structured, stable (half-life in theorder of 30 min) and inaccessible to either ribosome initiationor Sok-RNA binding. Slow 3

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples.