No abstract

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.

Complex biological processes are often regulated, at least in part, by the binding of transcription factors to their targets. Recently, considerable effort has been made to analyze the binding of relevant factors to the suite of targets they regulate, thereby generating a regulatory circuit map. However, for most studies the dynamics of binding have not been analyzed, and thus the temporal order of events and mechanisms by which this occurs are poorly understood. We globally analyzed in detail the temporal order of binding of several key factors involved in the salt response of yeast to their target genes. Analysis of Yap4 and Sko1 binding to their target genes revealed multiple temporal classes of binding patterns: (1) constant binding, (2) rapid induction, (3) slow induction, and (4) transient induction. These results demonstrate that individual transcription factors can have multiple binding patterns and help define the different types of temporal binding patterns used in eukaryotic gene regulation. To investigate these binding patterns further, we also analyzed the binding of seven other key transcription factors implicated in osmotic regulation, including Hot1, Msn1, Msn2, Msn4, Skn7, and Yap6, and found significant coassociation among the different factors at their gene targets. Moreover, the binding of several key factors was correlated with distinct classes of Yap4-and Sko1-binding patterns and with distinct types of genes. Gene expression studies revealed association of Yap4, Sko1, and other transcription factor-binding patterns with different gene expression patterns. The integration and analysis of binding and expression information reveals a complex dynamic and hierarchical circuit in which specific combinations of transcription factors target distinct sets of genes at discrete times to coordinate a rapid and important biological response.[Keywords: Salt response; stress; transcription factor; yeast] Supplemental material is available at http://www.genesdev.org.

Short (< 200 nt) RNA (sRNA) profiling of human cells using various technologies demonstrates unexpected complexity of sRNAs with 100's of thousands of sRNA species present 1, 2 , 3 , 4. Genetic and in vitro studies argue that these RNAs are not merely degradation products of longer transcripts but could indeed have function 1,2,5 . Furthermore, profiling of RNAs, including the sRNAs, can reveal not only novel transcripts, but also make clear predictions about the existence and properties of novel biochemical pathways operating in a cell. For example, short RNA profiling in human cells suggested existence of an unknown capping mechanism operating on cleaved RNA 2 a biochemical component of which was later identified 6 . Here we show that human cells contain a novel type of sRNAs that have non-genomically encoded 5' polyU tails. Presence of these RNAs at the termini of genes, specifically at the very 3' ends of known mRNAs strongly argues for the presence of a yet uncharacterized endogenous biochemical pathway in a cell that can copy RNA. We show that this pathway can operate on multiple genes, with specific enrichment towards transcripts encoding components of the translational machinery. Finally we show that genes are also flanked by sense, 3' polyadenylated sRNAs that are likely to be capped.To date, all sequencing reports characterizing sRNAs rely on conversion of RNA into cDNA employing ligation and/or amplification steps 7 . These steps could introduce bias in detecting

BackgroundDNA replication initiates at distinct origins in eukaryotic genomes, but the genomic features that define these sites are not well understood.ResultsWe have taken a combined experimental and bioinformatic approach to identify and characterize origins of replication in three distantly related fission yeasts: Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Using single-molecule deep sequencing to construct amplification-free high-resolution replication profiles, we located origins and identified sequence motifs that predict origin function. We then mapped nucleosome occupancy by deep sequencing of mononucleosomal DNA from the corresponding species, finding that origins tend to occupy nucleosome-depleted regions.ConclusionsThe sequences that specify origins are evolutionarily plastic, with low complexity nucleosome-excluding sequences functioning in S. pombe and S. octosporus, and binding sites for trans-acting nucleosome-excluding proteins functioning in S. japonicus. Furthermore, chromosome-scale variation in replication timing is conserved independently of origin location and via a mechanism distinct from known heterochromatic effects on origin function. These results are consistent with a model in which origins are simply the nucleosome-depleted regions of the genome with the highest affinity for the origin recognition complex. This approach provides a general strategy for understanding the mechanisms that define DNA replication origins in eukaryotes.