Biology subjects

Castello, A.

Publications and source records attributed to Castello, A..

26 records · Page 2Linked to original sources

bioRxiv2020

Proteome-wide Profiling of RNA-Binding Protein Responses to flg22 Reveals Novel Components of Plant Immunity

RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation and are known to contribute to plant immunity. To understand the responses of cellular RBPs to an immune elicitor, we applied RNA interactome capture to Arabidopsis leaves treated with flg22. Strikingly, flg22 induced a pervasive remodelling of the cellular RBPome affecting 186 proteins. Flg22-responsive RBPs included classical RBPs involved in RNA metabolism as well as non-canonical RBPs. RBP responders detected after 2h of treatment are enriched in putative sites for post-translational modifications, which may play a regulatory role. By contrast, changes in RBP abundance becomes increasingly important for the RBPome responses to flg22 after 12h. Plant resistance to Pseudomonas syringae is strongly altered in mutant lines lacking individual flg22-responsive RBPs, supporting the importance of RBP dynamics in plant immunity. This study provides a comprehensive and systematic census of flg22 responsive plant RBPs, discovering novel components of plant immunity.

molecular biology↗

bioRxiv2020

Comparative poly(A)+ RNA interactome capture of fission yeast RNA exosome mutants reveals insights into RNA biogenesis

The nuclear RNA exosome plays a key role in mediating degradation and processing of multiple cellular RNAs. Recognition of the specific RNA substrates by the exosome is mediated by the RNA-binding co-factors. Transient binding of co-factors either to the exosome or the substrate RNAs as well as their rapid decay make identification of the co-factors challenging. Here, we employ a comparative poly(A)+ RNA interactome capture approach in the fission yeast exosome mutants to identify proteins that interact with poly(A)+ RNA in an exosome-dependent manner. Our analyses identified multiple proteins whose occupancy on RNA is altered in the exosome mutants including zinc-finger protein Mub1. Mub1 is required to maintain the levels of a subset of the exosome RNA substrates including mRNAs encoding for stress-responsive proteins. Removal of the zinc finger domain leads to loss of RNA suppression under non-stressed conditions, altered expression of heat shock genes in response to stress, and reduced growth at elevated temperature. These findings highlight importance of exosome-dependent mRNA degradation in buffering gene expression networks to mediate cellular adaptation to stress.

genomics↗

bioRxiv2020

CryoSIM: super resolution 3D structured illumination cryogenic fluorescence microscopy for correlated ultra-structural imaging

Rapid cryo-preservation of biological specimens is the gold standard for visualising cellular structures in their true structural context. However, current commercial cryo-fluorescence microscopes are limited to low resolutions. To fill this gap, we have developed cryoSIM, a microscope for 3D super-resolution fluorescence cryo-imaging for correlation with cryo electron microscopy or cryo soft X-ray tomography. We provide the full instructions for replicating the instrument mostly from off-the-shelf components and accessible, user-friendly open source Python control software. Therefore, cryoSIM democratises the ability to detect molecules using super-resolution fluorescence imaging of cryo-preserved specimens for correlation with their cellular ultrastructure.

cell biology↗

bioRxiv2019

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

Production, function, and turnover of mRNA are orchestrated by multi-subunit machineries that play a central role in gene expression. Within these molecular machines, interactions with the target mRNA are mediated by RNA-binding proteins (RBPs), and the accuracy and dynamics of these RNA-protein interactions are essential for their function. Here, we show that fission yeast whole cell poly(A)+ RNA-protein crosslinking data provides system-wide information on the organisation and function of the RNA-protein complexes. We evaluate relative enrichment of cellular RBPs on poly(A)+ RNA to identify interactors with high RNA-binding activity and provide key information about the RNA-binding properties of large multi-protein complexes, such as the mRNA 3 end processing machinery (cleavage and polyadenylation factor, CPF) and the RNA exosome. We demonstrate that different functional modules within CPF differ in their ability to interact with RNA. Importantly, we reveal that CPF forms additional contacts with RNA via the Fip1 subunit of the polyadenylation module and two subunits of the nuclease module. In addition, our data highlights the central role of the RNA helicase Mtl1 in RNA degradation by the exosome as mutations in Mtl1 lead to disengagement of the exosome from RNA. We examine how routes of substrate access to the complex are affected upon mutation of exosome subunits. Our results provide important insights into how different components of the exosome contribute to engagement of the complex with substrate RNA. Overall, our data uncover how multi-subunit cellular machineries interact with RNA, on a proteome-wide scale.

molecular biology↗

bioRxiv2019

Identification of circular RNAs in porcine spermatozoa and their relation to sperm motility

Circular RNAs (circRNAs) are emerging as a novel class of noncoding RNAs which potential role as gene regulators is quickly gaining interest. Although circRNAs have been studied in several tissues and cell types across several animal species, the characterization of the circRNAome in ejaculated sperm remains unexplored. In this study, we profiled the sperm circRNA catalogue in 40 boar samples. A complex population of 1,598 circRNAs was shared in at least 30 samples. The predicted circRNAs presented in general low abundances and were highly tissue-specific. Circa 80% of the circRNAs identified in the boar sperm were reported as novel. We also constructed a circRNA-miRNA interaction network based on experimental and predictive microRNA (miRNA) binding sites. Moreover, we found significant correlation between the abundance of some circRNAs and sperm motility parameters and confirmed two of these correlations by RT-qPCR in 36 samples with extreme sperm quality. Our study provides a thorough characterization of a novel cell type for the circRNA encyclopedia collection and suggests that circRNAs are potential noninvasive biomarkers for male sperm quality and thus, might also hold potential to predict male fertility.

genomics↗

bioRxiv2018

A thorough RNA-seq characterization of the porcine sperm transcriptome and its seasonal changes

Understanding the molecular basis of cell function and ultimate phenotypes is crucial for the development of biological markers. With this aim, several RNA-seq studies have been devoted to characterize the transcriptome of ejaculated spermatozoa in relation to sperm quality and fertility. Semen quality follows a seasonal pattern and decays in the summer months in several animal species. The aim of this study was to deeply profile the transcriptome of the boar sperm and to evaluate its seasonal changes. We sequenced the total and the short fractions of the sperm RNA from 10 Pietrain boars, 5 collected in summer and 5 five sampled in winter, and identified a complex and rich transcriptome with 4,436 coding genes of moderate to high abundance. Transcript fragmentation was high but less obvious in genes related to spermatogenesis, chromatin compaction and fertility. Short non-coding RNAs mostly included piwi-interacting RNAs, transfer RNAs and micro-RNAs. We also compared the transcriptome of the summer and the winter ejaculates and identified 34 coding genes and 7 micro-RNAs with a significantly distinct distribution. These genes were mostly related to oxidative stress, DNA damage and autophagy. This is the deepest characterization of the boar sperm transcriptome and the first study linking the transcriptome and the seasonal variability of semen quality in animals. The annotation described here can be used as a reference for the identification of markers of sperm quality in pigs.

genomics↗

bioRxiv2018

Understanding RNP remodelling uncovers RBPs functionally required for viral replication

The compendium of RNA-binding proteins (RBPome) has been greatly expanded by the development of RNA-interactome capture (RNA-IC). However, it remains unknown how responsive is the RBPome and whether these responses are biologically relevant. To answer these questions, we created comparative RNA-IC to analyse cells challenged with an RNA virus, called sindbis (SINV). Strikingly, the virus altered the activity of 245 RBPs, many of which were newly discovered by RNA-IC. Mechanistically, alterations in RNA binding upon SINV infection are caused by changes in the subcellular localisation of RBPs and RNA availability. Moreover, RBPome responses are crucial, as perturbation of dynamic RBPs modulates the capacity of the virus to infect the cell. For example, ablation of XRN1 causes cells to be refractory to infection, while GEMIN5 moonlights as a novel antiviral factor. Therefore, RBPome remodelling provides a mechanism by which cells can extensively rewire gene expression in response to physiological cues.\n\nHIGHLIGHTSO_LIA quarter of the RBPome remodels upon SINV infection.\nC_LIO_LIThe remodelling is caused by changes in protein localisation and RNA availability.\nC_LIO_LIRewiring of the RBPome is crucial for viral infection efficacy.\nC_LIO_LIWe discover RBPs with previously unknown anti- or pro-viral activity.\nC_LI

molecular biology↗

bioRxiv2017

The small non-coding vault RNA1-1 acts as a riboregulator of autophagy

Vault RNAs (vtRNA) are small, 88-100nt non-coding RNAs found in many eukaryotes. Although they have been linked to drug resistance, apoptosis and nuclear transport, their function remains unclear. Here we show that a human vtRNA, RNA1-1, specifically binds to the autophagy receptor sequestosome-1/p62. Antisense-mediated depletion of vault RNA1-1 augments, whereas increased vault RNA1-1 expression restricts, autophagic flux in a p62-dependent manner. Bulk autophagy induced by starvation reduces the levels of vault RNA1-1 and the fraction of RNA-bound p62. These findings show that RNAs can act as riboregulators of biological processes by interacting with proteins, and assign a function to a vault RNA.

molecular biology↗