Single-cell proteo-genomic reveals a comprehensive map of centrosome-associated spliceosome components.

Ribonucleoprotein (RNP) condensates are crucial for controlling RNA metabolism and splicing events in animal cells. We used spatial proteomics and transcriptomic to elucidate RNP interaction networks at the centrosome, the main microtubule-organizing center in animal cells. We found a number of cell-type specific centrosome-associated spliceosome interactions localized in subcellular structures involved in nuclear division and ciliogenesis. A component of the nuclear spliceosome BUD31 was validated as an interactor of the centriolar satellite protein OFD1. Analysis of normal and disease cohorts identified the cholangiocarcinoma as target of centrosome-associated spliceosome alterations. Multiplexed single-cell fluorescent microscopy for the centriole linker CEP250 and spliceosome components including BCAS2, BUD31, SRSF2 and DHX35 recapitulated bioinformatic predictions on the centrosome-associated spliceosome components tissue-type specific composition. Collectively, centrosomes and cilia act as anchor for cell-type specific spliceosome components, and provide a helpful reference for explore cytoplasmic condensates functions in defining cell identity and in the origin of rare diseases.

New insights into the centrosome-associated spliceosome components as regulators of ciliogenesis and tissue identity.

Biomolecular condensates are membrane-less assemblies of proteins and nucleic acids. Centrosomes are biomolecular condensates that play a crucial role in nuclear division, cytoskeletal remodeling, and cilia formation in animal cells. Spatial omics technology is providing new insights into the dynamic exchange of spliceosome components between the nucleus and the centrosome/cilium. Intriguingly, centrosomes are emerging as cytoplasmic sites for information storage, enriched with RNA molecules and RNA-processing proteins. Furthermore, growing evidence supports the view that nuclear spliceosome components assembled at the centrosome function as potential coordinators of splicing subprograms, pluripotency, and cell differentiation. In this article, we first discuss the current understanding of the centrosome/cilium complex, which controls both stem cell differentiation and pluripotency. We next explore the molecular mechanisms that govern splicing factor assembly and disassembly around the centrosome and examine how RNA processing pathways contribute to ciliogenesis. Finally, we discuss numerous unresolved compelling questions regarding the centrosome-associated spliceosome components and transcript variants within the cytoplasm as sources of RNA-based secondary messages in the regulation of cell identity and cell fate determination. This article is categorized under: RNA-Based Catalysis > RNA Catalysis in Splicing and Translation RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Processing.

In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2.

Cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is a prominent example within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles, and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis, interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here, we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2. We also describe pH-dependent conformational changes of BicD2 using cryoelectron microscopy (cryo-EM), template-free structure predictions, and biophysical tools. Our results will help define the biochemical parameters for the in vitro reconstitution of higher-order BicD2 protein complexes.

Cryo-Electron Tomography and Proteomics studies of centrosomes from differentiated quiescent thymocytes.

We have used cryo Electron Tomography, proteomics and immunolabeling to study centrosomes isolated from the young lamb thymus, an efficient source of quiescent differentiated cells. We compared the proteome of thymocyte centrosomes to data published for KE37 cells, focusing on proteins associated with centriole disengagement and centrosome separation. The data obtained enhances our understanding of the protein system joining the centrioles, a system comprised of a branched network of fibers linked to an apparently amorphous density that was partially characterized here. A number of proteins were localized to the amorphous density by immunolabeling (C-NAP1, cohesin SMC1, condensin SMC4 and NCAPD2), yet not DNA. In conjuction, these data not only extend our understanding of centrosomes but they will help refine the model that focus on the protein system associated with the centriolar junction.

The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1.

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

Structural basis for the PufX-mediated dimerization of bacterial photosynthetic core complexes.

In Rhodobacter (Rba.) sphaeroides, the subunit PufX is involved in the dimeric organization of the core complex. Here, we report the 3D reconstruction at 12 A by cryoelectron microscopy of the core complex of Rba. veldkampii, a complex of approximately 300 kDa without symmetry. The core complex is monomeric and constituted by a light-harvesting complex 1 (LH1) ring surrounding a uniquely oriented reaction center (RC). The LH1 consists of 15 resolved alpha/beta heterodimers and is interrupted. Within the opening, PufX polypeptide is assigned at a position facing the Q(B) site of the RC. This core complex is different from a dissociated dimer of the core complex of Rba. sphaeroides revealing that PufX in Rba. veldkampii is unable to dimerize. The absence in PufX of Rba. veldkampii of a G(31)XXXG(35) dimerization motif highlights the transmembrane interactions between PufX subunits involved in the dimerization of the core complexes of Rhodobacter species.

Functional and structural analysis of the photosynthetic apparatus of Rhodobacter veldkampii.

In the widely studied purple bacterium Rhodobacter sphaeroides, a small transmembrane protein, named PufX, is required for photosynthetic growth and is involved in the supramolecular dimeric organization of the core complex. We performed a structural and functional analysis of the photosynthetic apparatus of Rhodobacter veldkampii, a related species which evolved independently. Time-resolved optical spectroscopy of R. veldkampii chromatophores showed that the reaction center shares with R. sphaeroides spectral and redox properties and interacts with a cytochrome bc(1) complex through a Q-cycle mechanism. Kinetic analysis of flash-induced cytochrome b(561) reduction indicated a fast delivery of the reduced quinol produced by the reaction center to the cytochrome bc(1) complex. A core complex, along with two light-harvesting LH2 complexes significantly different in size, was purified and analyzed by sedimentation, size exclusion chromatography, mass spectroscopy, and electron microscopy. A PufX subunit identified by MALDI-TOF was found to be associated with the core complex. However, as shown by sedimentation and single-particle analysis by electron microscopy, the core complex is monomeric, suggesting that in R. veldkampii, PufX is involved in the photosynthetic growth but is unable to induce the dimerization of the core complex.

Membrane insertion of Rhodopseudomonas acidophila light harvesting complex 2 investigated by high resolution AFM.

Light harvesting complexes 2 (LH2) are the peripheral antenna proteins in the bacterial photosynthetic apparatus and are built of alpha/beta-heterodimers containing three bacteriochlorophylls and two carotenoids each. Previously, we have found in 2D-crystals that the complexes could be inserted within the membrane with a tilt with respect to the membrane plane (Rhodobacter sphaeroides) or without tilt (Rubrivivax gelatinosus). To investigate whether the tilted insertion represents the native state or if it is due to specific 2D-crystal contacts, we have used atomic force microscopy to investigate LH2 from Rhodopseudomonas acidophila reconstituted at different lipid to protein ratios. High-resolution topographs could be acquired of two types of 2D-crystals or of densely packed membranes. Interestingly, in type 2 2D-crystals and in non-crystalline densely packed membranes, cylinders are integrated with their symmetry axis normal to the membrane plane, while in type 1 2D-crystals LH2 cylinders are integrated with a tilt of approximately 4 degrees with respect to the membrane plane. Therefore, we present strong evidence that the tilt of LH2 does not represent the native membrane state and is due to protein-protein contacts in specific 2D-crystals.

Structure of the dimeric PufX-containing core complex of Rhodobacter blasticus by in situ atomic force microscopy.

We have studied photosynthetic membranes of wild type Rhodobacter blasticus, a closely related strain to the well studied Rhodobacter sphaeroides, using atomic force microscopy. High-resolution atomic force microscopy topographs of both cytoplasmic and periplasmic surfaces of LH2 and RC-LH1-PufX (RC, reaction center) complexes were acquired in situ. The LH2 is a nonameric ring inserted into the membrane with the 9-fold axis perpendicular to the plane. The core complex is an S-shaped dimer composed of two RCs, each encircled by 13 LH1 alpha/beta-heterodimers, and two PufXs. The LH1 assembly is an open ellipse with a topography-free gap of approximately 25 A. The two PufXs, one of each core, are located at the dimer center. Based on our data, we propose a model of the core complex, which provides explanation for the PufX-induced dimerization of the Rhodobacter core complex. The QB site is located facing a approximately 25-A wide gap within LH1, explaining the PufX-favored quinone passage in and out of the core complex.

Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides.

Monomeric and dimeric PufX-containing core complexes have been purified from membranes of wild-type Rhodobacter sphaeroides. Reconstitution of both samples by detergent removal in the presence of lipids leads to the formation of two-dimensional crystals constituted of dimeric core complexes. Two-dimensional crystals were further analyzed by cryoelectron microscopy and atomic force microscopy. A projection map at 26-A resolution reveals that core complexes assemble in an "S"-shaped dimeric complex. Each core complex is composed of one reaction center, 12 light-harvesting 1 alpha/beta-heterodimers, and one PufX protein. The light-harvesting 1 assemblies are open with a gap of density of approximately 30-A width and surround oriented reaction centers. A maximum density is found at the dimer junction. Based on the projection map, a model is proposed, in which the two PufX proteins are located at the dimer junction, consistent with the finding of dimerization of monomeric core complexes upon reconstitution. This localization of PufX in the core complex implies that PufX is the structural key for the dimer complex formation rather than a channel-forming protein for the exchange of ubiquinone/ubiquinol between the reaction center and the cytochrome bc1 complex.