Specific Features of RNA Polymerases I and III: Structure and Assembly

RNA polymerase I (RNAPI) and RNAPIII are multi-heterogenic protein complexes that specialize in the transcription of highly abundant non-coding RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA). In terms of subunit number and structure, RNAPI and RNAPIII are more complex than RNAPII that synthesizes thousands of different mRNAs. Specific subunits of the yeast RNAPI and RNAPIII form associated subcomplexes that are related to parts of the RNAPII initiation factors. Prior to their delivery to the nucleus where they function, RNAP complexes are assembled at least partially in the cytoplasm. Yeast RNAPI and RNAPIII share heterodimer Rpc40-Rpc19, a functional equivalent to the αα homodimer which initiates assembly of prokaryotic RNAP. In the process of yeast RNAPI and RNAPIII biogenesis, Rpc40 and Rpc19 form the assembly platform together with two small, bona fide eukaryotic subunits, Rpb10 and Rpb12. We propose that this assembly platform is co-translationally seeded while the Rpb10 subunit is synthesized by cytoplasmic ribosome machinery. The translation of Rpb10 is stimulated by Rbs1 protein, which binds to the 3′-untranslated region of RPB10 mRNA and hypothetically brings together Rpc19 and Rpc40 subunits to form the αα-like heterodimer. We suggest that such a co-translational mechanism is involved in the assembly of RNAPI and RNAPIII complexes.

Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis

Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.

Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION

Macromolecular complexes that exhibit continuous forms of structural flexibility pose a challenge for many existing tools in cryo-EM single-particle analysis. We describe a new tool, called multi-body refinement, which models flexible complexes as a user-defined number of rigid bodies that move independently from each other. Using separate focused refinements with iteratively improved partial signal subtraction, the new tool generates improved reconstructions for each of the defined bodies in a fully automated manner. Moreover, using principal component analysis on the relative orientations of the bodies over all particles in the data set, we generate movies that describe the most important motions in the data. Our results on two test cases, a cytoplasmic ribosome from Plasmodium falciparum, and the spliceosomal B-complex from yeast, illustrate how multi-body refinement can be useful to gain unique insights into the structure and dynamics of large and flexible macromolecular complexes.Please note that this bioRxiv submission is ahead of the availability of the multi-body software in relion-3.0. We take great care in distributing stable software, but this does take time. We will announce the (beta-)release of relion-3.0 through the ccp-em mailing list (https://www.jiscmail.ac.uk/CCPEM) and on twitter (@SjorsScheres).

146 EFFECT OF PROTEIN SYNTHESIS INHIBITOR ON BOAR SPERM CAPACITATION AND FERTILIZATION IN VITRO

In human, bovine, mouse, and rat sperm, translation of RNA to proteins in the mitochondrial ribosome during capacitation has been reported to be important for fertilization. The objective of this study was to examine effect of protein synthesis inhibitor (ribosome inhibitor) on boar sperm capacitation and IVF. Sperm from an ejaculated sperm-rich fraction of Berkshire boars were washed by centrifugation (1500 rpm for 35 min) in a Percoll gradient (45/90%) and then incubated in modified Medium-199 containing 0.4% BSA and 5 mM caffeine sodium benzoate, supplemented with or without a mitochondrial ribosome-specific (55S ribosome) inhibitor, chloramphenicol (CP; 0.3 mM), or a cytoplasmic ribosome-specific (80S ribosome) inhibitor, cyclohexide (CH; 3.6 mM), in an atmosphere of 5% CO2 in air at 39°C for 45 or 90 min. At 45 and 90 min after culture, sperm viability, motility, and chlortetracyclin-stained patterns (to assess the sperm functional status, capacitation, and acrosome reaction) were examined. Porcine oocytes were matured in vitro for 44 h in porcine oocyte medium supplemented with eCG, hCG, and dibutyryl cyclic adenosine monophosphate for the first 20 h. Matured oocytes after the removal of cumulus cells were co-cultured with sperm (final conc.: 2.5 × 105 cells mL–1) in the absence or presence of CP or CH for 8 h. Sperm penetrability was also determined. Statistical analyses of data from 4 replicated trials were performed by ANOVA. After 45 and 90 min of culture, neither CP nor CH affected sperm viability and motility (P > 0.05). The addition of CP after 45 and 90 min of culture significantly (P < 0.05) decreased capacitated and acrosome-reacted sperm rates, as detected by chlortetracyclin fluorescence assay (capacitated: control 9.6 v. CP 5.6%, control 17.8 v. CP 10.2%; acrosome reacted: control 4.6 v. CP 2.2%, control 9.2 v. CP 4.8%, respectively; P < 0.05). In the presence of CH, IVF rate and number of sperm per penetrated egg were decreased (control 80.8 v. CH 46.8%, 2.2 v. 1.4, respectively; P < 0.05). In the presence of CH, however, the percentage of metaphase II oocytes after co-culture with sperm for 8 h was lower than other 2 groups (control 87.6 v. CP 85.5 v. CH 74.0%; P < 0.05), and the percentage of A/T-II oocytes was higher than in the other 2 groups (control 1.1 v. CP 0 v. CH 9.4%; P < 0.05). From these results, we conclude that mitochondrial ribosome-specific inhibitor, chloramphenicol, affects capacitation and acrosome reaction but not penetration, whereas cytoplasmic ribosome-specific inhibitor, cyclohexide, decreases the number of oocytes that reach metaphase II stage and are penetrated.

A new role for BiP

In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome–translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome–translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.