Structural basis of activation of the tumor suppressor protein neurofibromin.

Mutations in the NF1 gene cause the familial genetic disease neurofibromatosis type I, as well as predisposition to cancer. The NF1 gene product, neurofibromin, is a GTPase-activating protein and acts as a tumor suppressor by negatively regulating the small GTPase, Ras. However, structural insights into neurofibromin activation remain incompletely defined. Here, we provide cryoelectron microscopy (cryo-EM) structures that reveal an extended neurofibromin homodimer in two functional states: an auto-inhibited state with occluded Ras-binding site and an asymmetric open state with an exposed Ras-binding site. Mechanistically, the transition to the active conformation is stimulated by nucleotide binding, which releases a lock that tethers the catalytic domain to an extended helical repeat scaffold in the occluded state. Structure-guided mutational analysis supports functional relevance of allosteric control. Disease-causing mutations are mapped and primarily impact neurofibromin stability. Our findings suggest a role for nucleotides in neurofibromin regulation and may lead to therapeutic modulation of Ras signaling.

Modular assembly of the nucleolar pre-60S ribosomal subunit.

Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.

mTORC1 promotes TOP mRNA translation through site-specific phosphorylation of LARP1.

LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5'TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1's translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 'pendular hook' engages the TOP mRNA 5'-end to repress translation, but only in conditions of mTORC1 inhibition.

Assembly and early maturation of large subunit precursors.

The eukaryotic ribosome is assembled through a complex process involving more than 200 factors. As preribosomal RNA is transcribed, assembly factors bind the nascent pre-rRNA and guide its correct folding, modification, and cleavage. While these early events in the assembly of the small ribosomal subunit have been relatively well characterized, assembly of the large subunit precursors, or pre-60S, is less well understood. Recent structures of nucleolar intermediates of large subunit assembly have shed light on the role of many early large subunit assembly factors, but how these particles emerge is still unknown. Here, we use the expression and purification of truncated pre-rRNAs to examine the initial assembly of pre-60S particles. Using this approach, we can recapitulate the early recruitment of large subunit assembly factors mainly to the domains I, II, and VI of the assembling 25S rRNA.

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons.

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here 11 new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5'-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that 6 of 18 (33%) protein-coding mutations and the two (100%) 5'-UTR mutations display incomplete penetrance. Three mutations were identified in two independent kindreds, due to a hotspot or a founder effect. Finally, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

Assembly of the small ribosomal subunit in yeast: mechanism and regulation.

The eukaryotic ribosome is made of four intricately folded ribosomal RNAs and 79 proteins. During rapid growth, yeast cells produce an incredible 2000 ribosomes every minute. Ribosome assembly involves more than 200 trans-acting factors, intervening from the transcription of the preribosomal RNA in the nucleolus to late maturation events in the cytoplasm. The biogenesis of the small ribosomal subunit, or 40S, is especially intricate, requiring more than four times the mass of the small subunit in assembly factors for its full maturation. Recent studies have provided new insights into the complex assembly of the 40S subunit. These data from cryo-electron microscopy, X-ray crystallography, and other biochemical and molecular biology methods, have elucidated the role of many factors required in small subunit maturation. Mechanisms of the regulation of ribosome assembly have also emerged from this body of work. This review aims to integrate these new results into an updated view of small subunit biogenesis and its regulation, in yeast, from transcription to the formation of the mature small subunit.

Nucleolar maturation of the human small subunit processome.

The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryo­electron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome's impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.

Architecture of the yeast small subunit processome.

The small subunit (SSU) processome, a large ribonucleoprotein particle, organizes the assembly of the eukaryotic small ribosomal subunit by coordinating the folding, cleavage, and modification of nascent pre-ribosomal RNA (rRNA). Here, we present the cryo-electron microscopy structure of the yeast SSU processome at 5.1-angstrom resolution. The structure reveals how large ribosome biogenesis complexes assist the 5' external transcribed spacer and U3 small nucleolar RNA in providing an intertwined RNA-protein assembly platform for the separate maturation of 18S rRNA domains. The strategic placement of a molecular motor at the center of the particle further suggests a mechanism for mediating conformational changes within this giant particle. This study provides a structural framework for a mechanistic understanding of eukaryotic ribosome assembly in the model organism Saccharomyces cerevisiae.

The complete structure of the small-subunit processome.

The small-subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small-subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete near-atomic model of this assembly. In this nucleolar superstructure, 51 ribosome-assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry and steric hindrance, as well as protein- and RNA-mediated RNA remodeling, are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

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.

Stage-specific assembly events of the 6-MDa small-subunit processome initiate eukaryotic ribosome biogenesis.

Eukaryotic ribosome biogenesis involves a plethora of ribosome-assembly factors, and their temporal order of association with preribosomal RNA is largely unknown. By using Saccharomyces cerevisiae as a model organism, we developed a system that recapitulates and arrests ribosome assembly at early stages, thus providing in vivo snapshots of nascent preribosomal particles. Here we report the stage-specific order in which 70 ribosome-assembly factors associate with preribosomal RNA domains, thereby forming the 6-MDa small-subunit processome.

Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1.

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.