Andersson R.,Copenhagen University |
Refsing Andersen P.,Center for Biogenesis and Metabolism |
Valen E.,University of Bergen |
Valen E.,Harvard University |
And 7 more authors.
Nature Communications | Year: 2014
Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs. © 2014 Macmillan Publishers Limited.
Nielsen K.H.,University of Aarhus |
Nielsen K.H.,Center for Biogenesis and Metabolism |
Behrens M.A.,Center for Biogenesis and Metabolism |
Behrens M.A.,University of Aarhus |
And 11 more authors.
Nucleic Acids Research | Year: 2011
eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5kDa eIF4G fragment (eIF4G-MC), with a high-and a low-affinity site, having binding constants KD of ∼50 and ∼1000nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A-eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A-eIF4B-RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A. © 2011 The Author(s).