Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel

Pre-ribosomal particles evolve in the nucleus through transient interaction with biogenesis factors, before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle purified from Saccharomyces cerevisiae through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-electron microscopy reconstruction of the Arx1-particle at 11.9 Å resolution reveals regions of extra densities on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immuno-electron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation

UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes—UtpA and UtpB—interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA–protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5′ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5′ ETS and U3 snoRNA as well as the 3′ boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly

Mechanism of eIF6 release from the nascent 60S ribosomal subunit.

SBDS protein (deficient in the inherited leukemia-predisposition disorder Shwachman-Diamond syndrome) and the GTPase EFL1 (an EF-G homolog) activate nascent 60S ribosomal subunits for translation by catalyzing eviction of the antiassociation factor eIF6 from nascent 60S ribosomal subunits. However, the mechanism is completely unknown. Here, we present cryo-EM structures of human SBDS and SBDS-EFL1 bound to Dictyostelium discoideum 60S ribosomal subunits with and without endogenous eIF6. SBDS assesses the integrity of the peptidyl (P) site, bridging uL16 (mutated in T-cell acute lymphoblastic leukemia) with uL11 at the P-stalk base and the sarcin-ricin loop. Upon EFL1 binding, SBDS is repositioned around helix 69, thus facilitating a conformational switch in EFL1 that displaces eIF6 by competing for an overlapping binding site on the 60S ribosomal subunit. Our data reveal the conserved mechanism of eIF6 release, which is corrupted in both inherited and sporadic leukemias.Supported by a Federation of European Biochemical Societies Long term Fellowship (to FW), Specialist Programme from Bloodwise [12048] (AJW), the Medical Research Council [MC_U105161083] (AJW) and [U105115237] (RRK), Wellcome Trust strategic award to the Cambridge Institute for Medal Research [100140], Tesni Parry Trust (AJW), Ted’s Gang (AJW) and the Cambridge NIHR Biomedical Research Centre.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nsmb.311

parMERASA – multicore execution of parallelised hard real-time applications supporting analysability

Abstract-Engineers who design hard real-time embedded systems express a need for several times the performance available today while keeping safety as major criterion. A breakthrough in performance is expected by parallelizing hard real-time applications and running them on an embedded multi-core processor, which enables combining the requirements for high-performance with timing-predictable execution. parMERASA will provide a timing analyzable system of parallel hard real-time applications running on a scalable multicore processor. parMERASA goes one step beyond mixed criticality demands: It targets future complex control algorithms by parallelizing hard real-time programs to run on predictable multi-/many-core processors. We aim to achieve a breakthrough in techniques for parallelization of industrial hard real-time programs, provide hard real-time support in system software, WCET analysis and verification tools for multi-cores, and techniques for predictable multi-core designs with up to 64 cores

Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics

Gene networks are an efficient route for associating candidate genes with biological processes. Here, networks are used to discover more than 15 new genes for ribosomal subunit maturation, rRNA processing, and ribosomal export from the nucleus

A Protein Inventory of Human Ribosome Biogenesis Reveals an Essential Function of Exportin 5 in 60S Subunit Export

A systematic search for human ribosome biogenesis factors shows conservation of many aspects of eukaryotic ribosome synthesis with the well-studied process in yeast and identifies an export route of 60S subunits that is specific for higher eukaryotes

Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export

Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes (1-3). Nuclear export is a key, irreversible step in pre-ribosome maturation(4-8), but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved GTPase Nug2/Nog2 (called NGP-1, Gnl2 or nucleostemin 2 in human(9)) plays a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor(10). Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding prior to Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre-60S remodeling factors, the Rea1 ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre-60S particles requires both its K(+)-dependent GTPase activity and the remodeling ATPase activity of Rea1. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery

Die eigene Hochschule. Internationale Frauenuniversitaet 'Technik und Kultur'

SIGLEAvailable from TIB Hannover: F01B1124(3) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung, Berlin (Germany)DEGerman