Protein-Protein Interactions within Late Pre-40S Ribosomes

Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps

Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of the cytoplasmic translation initiation factor eIF5b/Fun12. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associate with Fun12 and form 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP-hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together

Eukaryotic Cells Producing Ribosomes Deficient in Rpl1 Are Hypersensitive to Defects in the Ubiquitin-Proteasome System

It has recently become clear that the misassembly of ribosomes in eukaryotic cells can have deleterious effects that go far beyond a simple shortage of ribosomes. In this work we find that cells deficient in ribosomal protein L1 (Rpl1; Rpl10a in mammals) produce ribosomes lacking Rpl1 that are exported to the cytoplasm and that can be incorporated into polyribosomes. The presence of such defective ribosomes leads to slow growth and appears to render the cells hypersensitive to lesions in the ubiquitin-proteasome system. Several genes that were reasonable candidates for degradation of 60S subunits lacking Rpl1 fail to do so, suggesting that key players in the surveillance of ribosomal subunits remain to be found. Interestingly, in spite of rendering the cells hypersensitive to the proteasome inhibitor MG132, shortage of Rpl1 partially suppresses the stress-invoked temporary repression of ribosome synthesis caused by MG132.United States. National Institutes of Health (GM25532)United States. National Institutes of Health (ARRAGM25532-S1)United States. National Institutes of Health (GM085177)United States. National Institutes of Health (CAI-3330)Natural Sciences and Engineering Research Council of Canada (NSERC

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

Splicing Endonuclease Is an Important Player in rRNA and tRNA Maturation in Archaea

In all three domains of life, tRNA genes contain introns that must be removed to yield functional tRNA. In archaea and eukarya, the first step of this process is catalyzed by a splicing endonuclease. The consensus structure recognized by the splicing endonuclease is a bulge-helix-bulge (BHB) motif which is also found in rRNA precursors. So far, a systematic analysis to identify all biological substrates of the splicing endonuclease has not been carried out. In this study, we employed CRISPRi to repress expression of the splicing endonuclease in the archaeon Haloferax volcanii to identify all substrates of this enzyme. Expression of the splicing endonuclease was reduced to 1% of its normal level, resulting in a significant extension of lag phase in H. volcanii growth. In the repression strain, 41 genes were down-regulated and 102 were up-regulated. As an additional approach in identifying new substrates of the splicing endonuclease, we isolated and sequenced circular RNAs, which identified excised introns removed from tRNA and rRNA precursors as well as from the 5 ' UTR of the gene HVO_1309. In vitro processing assays showed that the BHB sites in the 5 ' UTR of HVO_1309 and in a 16S rRNA-like precursor are processed by the recombinant splicing endonuclease. The splicing endonuclease is therefore an important player in RNA maturation in archaea

Incomplete lineage sorting and ancient admixture, and speciation without morphological change in ghost-worm cryptic species

Morphologically similar species, that is cryptic species, may be similar or quasi-similar owing to the deceleration of morphological evolution and stasis. While the factors underlying the deceleration of morphological evolution or stasis in cryptic species remain unknown, decades of research in the field of paleontology on punctuated equilibrium have originated clear hypotheses. Species are expected to remain morphologically identical in scenarios of shared genetic variation, such as hybridization and incomplete lineage sorting, or in scenarios where bottlenecks reduce genetic variation and constrain the evolution of morphology. Here, focusing on three morphologically similar Stygocapitella species, we employ a whole-genome amplification method (WGA) coupled with double-digestion restriction-site associated DNA sequencing (ddRAD) to reconstruct the evolutionary history of the species complex. We explore population structure, use population-level statistics to determine the degree of connectivity between populations and species, and determine the most likely demographic scenarios which generally reject for recent hybridization. We find that the combination of WGA and ddRAD allowed us to obtain genomic-level data from microscopic eukaryotes (∼1 millimetre) opening up opportunities for those working with population genomics and phylogenomics in such taxa. The three species share genetic variance, likely from incomplete lineage sorting and ancient admixture. We speculate that the degree of shared variation might underlie morphological similarity in the Atlantic species complex

Incomplete lineage sorting and ancient admixture, and speciation without morphological change in ghost-worm cryptic species

Morphologically similar species, that is cryptic species, may be similar or quasi-similar owing to the deceleration of morphological evolution and stasis. While the factors underlying the deceleration of morphological evolution or stasis in cryptic species remain unknown, decades of research in the field of paleontology on punctuated equilibrium have originated clear hypotheses. Species are expected to remain morphologically identical in scenarios of shared genetic variation, such as hybridization and incomplete lineage sorting, or in scenarios where bottlenecks reduce genetic variation and constrain the evolution of morphology. Here, focusing on three morphologically similar Stygocapitella species, we employ a whole-genome amplification method (WGA) coupled with double-digestion restriction-site associated DNA sequencing (ddRAD) to reconstruct the evolutionary history of the species complex. We explore population structure, use population-level statistics to determine the degree of connectivity between populations and species, and determine the most likely demographic scenarios which generally reject for recent hybridization. We find that the combination of WGA and ddRAD allowed us to obtain genomic-level data from microscopic eukaryotes (∼1 millimetre) opening up opportunities for those working with population genomics and phylogenomics in such taxa. The three species share genetic variance, likely from incomplete lineage sorting and ancient admixture. We speculate that the degree of shared variation might underlie morphological similarity in the Atlantic species complex