13 research outputs found
High-throughput RNA structure probing reveals critical folding events during early 60S ribosome assembly in yeast
While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast
Interrelationships between Yeast Ribosomal Protein Assembly Events and Transient Ribosome Biogenesis Factors Interactions in Early Pre-Ribosomes
Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains
In Vitro Reconstitution of Yeast tUTP/UTP A and UTP B Subcomplexes Provides New Insights into Their Modular Architecture
Enhanced FGF23 Serum Concentrations and Phosphaturia in Gene Targeted Mice Expressing WNK-Resistant Spak
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107752.pdf (publisher's version ) (Open Access)Background: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na(+),K(+),2Cl(-) cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. Methods: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak(tg/tg)) and in mice expressing wild type SPAK (spak(wt/wt)). Results: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spak(tg/tg) mice than in spak(wt/wt) mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spak(tg/tg) mice compared to spak(wt/wt) mice. Treatment of spak(wt/wt) mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. Conclusions: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca(2+) and phosphate excretion
U.S. policy in the UN environmental arena: powerful laggard or constructive leader?
Environment, Sustainable development, United Nations, United States,