Biology subjects

Spanos, C.

Publications and source records attributed to Spanos, C..

5 recordsLinked to original sources

bioRxiv2018

Defining the RNA Interactome by Total RNA-Associated Protein Purification

UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae) and bacteria (E. coli). In all analyses, SILAC labelling was used to quantify protein recovery in the presence and absence of irradiation. For S. cerevisiae, we also compared crosslinking using 254 nm (UVC) irradiation (TRAPP) with 4-thiouracil (4tU) labelling combined with ~350 nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was substantially higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its binding to tRNA loops in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation for highly abundant protein. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified in 395 peptides from 155 unique proteins, following phospho-peptide enrichment combined with a bioinformatics pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.

cell biology↗

bioRxiv2018

Fission yeast NDR/LATS kinase Orb6 regulates exocytosis via phosphorylation of exocyst complex

NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals, but few of their substrates are known. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity via spatial regulation of Gef1, a guanine nucleotide exchange factor for the small GTPase Cdc42. Here we show that Orb6 plays a critical role as a positive regulator of exocytosis, independent of Gef1. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify several proteins involved in membrane trafficking as Orb6 targets, and we confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro. Our results suggest that Orb6 kinase activity is crucial for exocyst localization to actively-growing cell tips and for exocyst activity during septum dissolution after cytokinesis. We further show that Orb6 phosphorylation of Sec3 serine-201 contributes to exocyst function in parallel with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.

cell biology↗

bioRxiv2017

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the {gamma}-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.

cell biology↗

bioRxiv2017

Swr1 mediated H2A.Z/Pht1 incorporation designates centromere DNA for de novo CENP-A/Cnp1 assembly

The underlying hallmark of centromeres is the presence of specialized nucleosomes in which histone H3 is replaced by CENP-A. The events that mediate the installation of CENP-A in place of H3 remain poorly characterized. H2A.Z is linked to transcriptional competence and associates with mammalian centromeres. We find that H2A.ZPht1 and the Swr1 complex are enriched in fission yeast CENP-ACnp1 chromatin. Our analysis shows that Swr1, Msc1 and H2A.ZPht1 are required to maintain CENP-ACnp1 chromatin integrity. Cell cycle analyses demonstrate that H2A.ZPht1 is deposited in S phase, coincident with the deposition of placeholder H3, and prior to CENP-ACnp1 replenishment in G2. Establishment assays reveal that H2A.ZPht1 and Swr1 are required for de novo assembly of CENP-ACnp1 onto naive centromere DNA. We propose that features akin to promoters within centromere DNA program the incorporation of H2A.ZPht1 via Swr1, and mediate the replacement of resident H3 nucleosomes with CENP-A nucleosomes thereby defining centromeres.

cell biology↗

bioRxiv2017

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. Almost nothing is known about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25s endogenous RNA targets and protein binding partners. Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase function. These results reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor for their biological functions.

molecular biology↗