Regulators of male and female sexual development are critical for the transmission of a malaria parasite.

Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.

Evolutionary compaction and adaptation visualized by the structure of the dormant microsporidian ribosome.

Microsporidia are eukaryotic parasites that infect essentially all animal species, including many of agricultural importance1-3, and are significant opportunistic parasites of humans4. They are characterized by having a specialized infection apparatus, an obligate intracellular lifestyle5, rudimentary mitochondria and the smallest known eukaryotic genomes5-7. Extreme genome compaction led to minimal gene sizes affecting even conserved ancient complexes such as the ribosome8-10. In the present study, the cryo-electron microscopy structure of the ribosome from the microsporidium Vairimorpha necatrix is presented, which illustrates how genome compaction has resulted in the smallest known eukaryotic cytoplasmic ribosome. Selection pressure led to the loss of two ribosomal proteins and removal of essentially all eukaryote-specific ribosomal RNA (rRNA) expansion segments, reducing the rRNA to a functionally conserved core. The structure highlights how one microsporidia-specific and several repurposed existing ribosomal proteins compensate for the extensive rRNA reduction. The microsporidian ribosome is kept in an inactive state by two previously uncharacterized dormancy factors that specifically target the functionally important E-site, P-site and polypeptide exit tunnel. The present study illustrates the distinct effects of evolutionary pressure on RNA and protein-coding genes, provides a mechanism for ribosome inhibition and can serve as a structural basis for the development of inhibitors against microsporidian parasites.

Conformational switches control early maturation of the eukaryotic small ribosomal subunit.

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5' external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5' external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5' external transcribed spacer ribonucleoprotein - provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

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.

Assembly and structure of the SSU processome-a nucleolar precursor of the small ribosomal subunit.

The small subunit processome is the first precursor of the small eukaryotic ribosomal subunit. During its assembly in the nucleolus, many ribosome biogenesis factors, an RNA chaperone, and ribosomal proteins associate with the nascent pre-rRNA. Biochemical studies have elucidated the rRNA-subdomain dependent recruitment of these factors during SSU processome assembly and have been complemented by structural studies of the assembled particle. Ribosome biogenesis factors encapsulate and guide subdomains of pre-ribosomal RNA in distinct compartments. This prevents uncoordinated maturation and enables processing of regions not accessible in the mature subunit. By sequentially reducing conformational freedom, flexible proteins facilitate the incorporation of dynamic subcomplexes into a globular particle. Large rearrangements within the SSU processome are required for compaction into the mature small ribosomal subunit.

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.

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.

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.