Gateways to the FANTOM5 promoter level mammalian expression atlas

The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource (http://fantom.gsc.riken.jp/5/). This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0560-6) contains supplementary material, which is available to authorized users

Cloning of RNA molecules in vitro.

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Q beta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro

Spontaneous rearrangements in RNA sequences

AbstractThe ability of RNAs to spontaneously rearrange their sequences under physiological conditions is demonstrated using the molecular colony technique, which allows single RNA molecules to be detected provided that they are amplifiable by the replicase of bacteriophage Qβ. The rearrangements are Mg2+-dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10−9 h−1 per site. The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of Qβ replicase, and have a number of biologically important implications

Structural basis for RNA-genome recognition during bacteriophage Q replication

Upon infection of Escherichia coli by bacteriophage Q, the virus-encoded -subunit recruits host trans-lation elongation factors EF-Tu and EF-Ts and riboso-mal protein S1 to form the Q replicase holoenzyme complex, which is responsible for amplifying the Q (+)-RNA genome. Here, we use X-ray crystallogra-phy, NMR spectroscopy, as well as sequence con-servation, surface electrostatic potential and muta-tional analyses to decipher the roles of the -subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1–2) in the recognition of Q (+)-RNA by the Q replicase complex. We show how three basic residues of the subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Q replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the -subunit and protein S1 cooperatively bind the (+)-stranded Q genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation