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Spatial regulation of RNA plays a critical role in gene expression regulation and cellular function. Understanding spatially resolved RNA dynamics and translation is vital for bringing new insights into biological processes such as embryonic development, neurobiology, and disease pathology. This review explores past studies in subcellular, cellular, and tissue-level spatial RNA biology driven by diverse methodologies, ranging from cell fractionation, in situ and proximity labeling, imaging, spatially indexed next-generation sequencing (NGS) approaches, and spatially informed computational modeling. Particularly, recent advances have been made for near-genome-scale profiling of RNA and multimodal biomolecules at high spatial resolution. These methods enabled new discoveries into RNA's spatiotemporal kinetics, RNA processing, translation status, and RNA-protein interactions in cells and tissues. The evolving landscape of experimental and computational strategies reveals the complexity and heterogeneity of spatial RNA biology with subcellular resolution, heralding new avenues for RNA biology research.

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Conformational diseases, such as Alzheimer's, Parkinson's and Huntington's diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington's disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. There is still no successful strategy to treat HD. In addition, the current understanding of the biological processes leading to the oligomerization and aggregation of proteins bearing the poly-Q tract has been derived from studies conducted on isolated poly-Q monomers and oligomers, whose structural properties are still unclear and often inconsistent. Here we describe a standardised biochemical approach to analyse by isopycnic ultracentrifugation the oligomerization of the N-terminal fragment of mutant HTT. The dynamic range of our method allows one to detect large and heterogeneous HTT complexes. Hence, it could be harnessed for the identification of novel molecular determinants responsible for the aggregation and the prion-like spreading properties of HTT in the context of HD. Equally, it provides a tool to test novel small molecules or bioactive compounds designed to inhibit the aggregation of mutant HTT.

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Plant viruses depend on host cellular factors for their replication and movement. There are cellular proteins that change their localization and/or expression and have a proviral role or antiviral activity and interact with or target viral proteins. Identification of those proteins and their roles during infection is crucial for understanding plant-virus interactions and to design antiviral resistance in crops. Important host proteins have been identified using approaches such as tag-dependent immunoprecipitation or yeast two hybridization that require cloning individual proteins or the entire virus. However, the number of possible interactions between host and viral proteins is immense. Therefore, an alternative method is needed for proteome-wide identification of host proteins involved in host-virus interactions. Here, we present cell fractionation coupled with mass spectrometry as an option to identify protein-protein interactions between viruses and their hosts. This approach involves separating subcellular organelles using differential and/or gradient centrifugation from virus-free and virus-infected cells (1) followed by comparative analysis of the proteomic profiles obtained for each subcellular organelle via mass spectrometry (2). After biological validation, prospect host proteins with proviral or antiviral roles can be subject to fundamental studies in the context of basic biology to shed light on both virus replication and cellular processes. They can also be targeted via gene editing to develop virus-resistant crops.

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Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5'-hydroxyl and 2'/3'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions.

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G1P3/IFI6 is an interferon stimulated gene with antiapoptotic, prometastatic, and antiviral functions. Despite its pleiotropic functions, subcellular localization of G1P3 remains unclear. Using biochemical- and confocal microscopic approaches, this study identified the localization of G1P3 in organelles of the endomembrane system and in the mitochondria of breast cancer cells. In cell fractionation studies, both interferon-induced endogenous- and stably expressed G1P3 cofractionated with affinity-isolated mitochondria. Results of the protease protection assay have suggested that ~24% of mitochondrial G1P3 resides within the mitochondria. Conforming to this, confocal microscopy studies of cells stably expressing epitope-tagged G1P3 (MCF-7/G1P3-FLAG), identified its localization in mitochondria (~38%) as well as in ER, trans-Golgi network (TGN), lysosomes, and in RAB5 positive (RAB5+ ) endosomes. These results suggested the trafficking of G1P3 from TGN into endolysosomes. Both G1P3 and RAB5 were known to confer apoptosis resistance through mitochondrial stabilization. Therefore, the effects of G1P3 on the localization of RAB5 in mitochondria were tested. Compared to vector control, the co-occurrence of RAB5 with the mitochondria was increased by 1.5-fold in MCF-7/G1P3-FLAG expressing cells (p ≤ .005). Taken together, our results demonstrate a role for G1P3 to promote the association of RAB5+ endosomes with mitochondria and provide insight into yet another mechanism of G1P3-induced cancer cell survival.

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Across a variety of systems, thousands of RNAs are localized to specific subcellular locations. However, for the vast majority of these RNAs, the mechanisms that underlie their transport are unknown. Historically, these mechanisms were uncovered for a single transcript at a time by laboriously testing the ability of RNA fragments to direct transcript localization. Recently developed methods profile the content of subcellular transcriptomes using high-throughput sequencing, allowing the analysis of the localization of thousands of transcripts at once. By identifying commonalities shared among multiple localized transcripts, these methods have the potential to rapidly expand our understanding of RNA localization mechanisms.

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Ribosome biogenesis is one cell function-defining process. It depends on efficient transcription of rDNAs in the nucleolus as well as on the cytosolic synthesis of ribosomal proteins. For newly transcribed rRNA modification and ribosomal protein assembly, so-called small nucleolar RNAs (snoRNAs) and ribosome biogenesis factors (RBFs) are required. For both, an inventory was established for model systems like yeast and humans. For plants, many assignments are based on predictions. Here, RNA deep sequencing after nuclei enrichment was combined with single molecule species detection by northern blot and in vivo fluorescence in situ hybridization (FISH)-based localization studies. In addition, the occurrence and abundance of selected snoRNAs in different tissues were determined. These approaches confirm the presence of most of the database-deposited snoRNAs in cell cultures, but some of them are localized in the cytosol rather than in the nucleus. Further, for the explored snoRNA examples, differences in their abundance in different tissues were observed, suggesting a tissue-specific function of some snoRNAs. Thus, based on prediction and experimental confirmation, many plant snoRNAs can be proposed, while it cannot be excluded that some of the proposed snoRNAs perform alternative functions than are involved in rRNA modification.

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Protein subcellular localization is an essential and highly regulated determinant of protein function. Major advances in mass spectrometry and imaging have allowed the development of powerful spatial proteomics approaches for determining protein localization at the whole cell scale. Here, a brief overview of current methods is presented, followed by a detailed discussion of organellar mapping through proteomic profiling. This relatively simple yet flexible approach is rapidly gaining popularity, because of its ability to capture the localizations of thousands of proteins in a single experiment. It can be used to generate high-resolution cell maps, and as a tool for monitoring protein localization dynamics. This review highlights the strengths and limitations of the approach and provides guidance to designing and interpreting profiling experiments.

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Eukaryotic flagella are conserved multifunctional organelles with roles in motility, intercellular interactions, and signal transduction. Leishmania possess a single flagellum at all stages of their life cycle. Flagella of promastigote forms in the fly are long and motile, with a canonical 9 + 2 microtubule axoneme and an extra-axonemal paraflagellar rod (PFR). This protocol describes a simple method for the isolation of Leishmania mexicana promastigote flagella, optimized to yield intact flagella that retain both the cytoskeletal elements (9 + 2 axoneme and PFR) and the surrounding membrane. The isolated flagella and deflagellated cell bodies are suitable for analysis by electron microscopy, protein mass spectrometry, and lipidomics.

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Antibiotics with new modes of action that are active against intracellular forms of Staphylococcus aureus are sorely needed to fight recalcitrant infections caused by this bacterium. Afabicin desphosphono (Debio 1452, the active form of afabicin [Debio 1450]) is an inhibitor of FabI enoyl-Acyl carrier protein reductase and has specific and extremely potent activity against Staphylococci, including strains resistant to current antistaphylococcal agents. Using mouse J774 macrophages and human THP-1 monocytes, we showed that afabicin desphosphono: (i) accumulates rapidly in cells, reaching stable cellular-to-extracellular concentration ratios of about 30; (ii) is recovered entirely and free in the cell-soluble fraction (no evidence of stable association with proteins or other macromolecules). Afabicin desphosphono caused a maximum cfu decrease of about 2.5 log10 after incubation in broth for 30 h, including against strains resistant to vancomycin, daptomycin, and/or linezolid. Using a pharmacodynamic model of infected THP-1 monocytes (30 h of incubation post-phagocytosis), we showed that afabicin desphosphono is bacteriostatic (maximum cfu decrease: 0.56 to 0.73 log10) towards all strains tested, a behaviour shared with the comparators (vancomycin, daptomycin, and linezolid) when tested against susceptible strains. We conclude that afabicin desphosphono has a similar potential as vancomycin, daptomycin or linezolid to control the intracellular growth and survival of phagocytized S. aureus and remains fully active against strains resistant to these comparators.

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Male infertility is widespread and estimated to affect 1 in 20 men. Although in some cases the etiology of the condition is well understood, for at least 50% of men, the underlying cause is yet to be classified. Male infertility, or subfertility, is often diagnosed by looking at total sperm produced, motility of the cells and overall morphology. Although counting spermatozoa and their associated motility is routine, morphology assessment is highly subjective, mainly because of the procedure being based on microscopic examination. A failure to diagnose male-infertility or sub-fertility has led to a situation where assisted conception is often used unnecessarily. As such, biomarkers of male infertility are needed to help establish a more consistent diagnosis. In the present study, we compared nuclear extracts from both high- and low-quality spermatozoa by LC-MS/MS based proteomic analysis. Our data shows that nuclear retention of specific proteins is a common facet among low-quality sperm cells. We demonstrate that the presence of Topoisomerase 2A in the sperm head is highly correlated to poor head morphology. Topoisomerase 2A is therefore a potential new biomarker for confirming male infertility in clinical practice.

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Cellular organelle fractionation, a basic technique in molecular biology, has been devised to separate various cell components, which can then be purified and analyzed biochemically. Isolation of proteins or RNAs from these fractions provides insight into fraction-specific or even organelle-specific expression, which may indicate potential modes of functionality or likelihood for a transcript to encode a protein. These findings can be further utilized to observe differences in expression between normal and diseased cell states, such as cancer. We utilize these techniques to observe expression of chimeric RNAs in these fractions. Within this chapter we describe the most frequently used cellular fractionation technique: the separation of the cytoplasmic fraction from the nuclear fraction in a cell.

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RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.

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Starch is one of the main carbohydrates in food; it is formed by two polysaccharides: amylose and amylopectin. The granule size of starch varies with different botanical origins and ranges from less than 1 µm to more than 100 µm. Some physicochemical and functional properties vary with the size of the granule, which makes it of great interest to find an efficient and accurate size-based separation method. In this study, the full-feed depletion mode of split-flow thin cell fractionation (FFD-SF) was employed for a size-based fractionation of two types of starch granules (corn and potato) on a large scale. The fractionation efficiency (FE) of fraction-a for corn and potato granules was 98.4 and 99.4%, respectively. The FFD-SF fractions were analyzed using optical microscopy (OM) and gravitational field-flow fractionation (GrFFF). The respective size distribution results were in close agreement for the corn starch fractions, while they were slightly different for the potato starch fractions. The thermal properties of FFD-SF fractions were analyzed, and the results for the potato starch showed that the peak temperature of gelatinization (Tp) slightly decreases as the size of the granules increases. Additionally, the enthalpy of gelatinization (ΔH) increases when the granule size increases and shows negative correlation with the gelatinization range (ΔT).

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The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

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In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as ß-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite's glycosomes.

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Homeostasis and the complex functions of organisms and cells rely on the sophisticated spatial and temporal regulation of signaling in different intra- and extracellular compartments and via different mediators. We here present a set of fast and easy to use protocols for the target-specific immunomagnetic enrichment of receptor containing endosomes (receptosomes), plasma membranes, lysosomes and exosomes. Isolation of subcellular organelles and exosomes is prerequisite for and will advance their detailed subsequent biochemical and functional analysis. Sequential application of the different subprotocols allows isolation of morphological and functional intact organelles from one pool of cells. The enrichment is based on a selective labelling using receptor ligands or antibodies together with superparamagnetic microbeads followed by separation in a patented matrix-free high-gradient magnetic purification device. This unique magnetic chamber is based on a focusing system outside of the empty separation column, generating an up to 3 T high-gradient magnetic field focused at the wall of the column.

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The growth and the survival of the human malaria parasite Plasmodium falciparum are critically dependent on the functions of the two organelles - the mitochondrion and the apicoplast. However, these two organelles have been known to be difficult to separate from each other when they are released from Plasmodium cell. We have been searching for the conditions with which separation of the mitochondrion and the apicoplast is achieved. In this study, we investigated how the two organelle's separation is affected when the pressure of the nitrogen gas to disrupt the Plasmodium cells by nitrogen cavitation method is lowered from the pressure regularly applied (1200 psi). The parasite cell was sufficiently disrupted even when nitrogen cavitation was carried out at 300 psi. The obtained mitochondrial sample was much less contaminated by DNA compared with the sample prepared using the gas at the regular pressure. After the fractionation by Percoll density gradient, the mitochondrion and the apicoplast from the 300 psi cell lysate exhibited different separation profiles. This is the first experimental evidence that indicates the mitochondrion and the apicoplast of P. falciparum are separable from each other.

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Necroptosis is a caspase-independent form of programmed cell death that is induced by a variety of different signalling cascades-all culminating in the activation of the pseudokinase mixed lineage kinase domain-like (MLKL). TNF-induced necroptosis is the most intensively studied of these pathways. Here we describe reagents and cell-based techniques that can be used to investigate TNF-mediated necroptosis in the lab.

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Studies conducted in the past decade have reported nucleostemin (NS) as a nucleolar protein that has a role in self-renewal and cell cycle regulation in cancer/stem cells, but is absent in differentiated cells. The localization and expression patterns of NS have always been disputed, as reports indicate its varied levels among tissues and cells. This study evaluates the expression and localization pattern of NS in normal cells, cancer cell lines, and stem cells. Our findings revealed that the expression of NS was high in cancers originating from the skin and liver compared to the normal cell lines. NS knockdown effects the proliferation of normal cell lines, similar to cancerous cell lines. The localization pattern of NS was analyzed by immunofluorescence, which showed that NS was localized in the nuclei of normal cell lines but is present both in the nucleus and the cytoplasm of cancerous/stem cell lines. Interestingly, we observed that siNS cancerous cell lines had lower NS in the cytoplasm, which did not salvage the reduction in proliferation caused by siNS. We postulate that the loss of NS in the nucleus inhibits the proliferative ability of both normal and cancerous cells at similar rates, although the role of NS in the cytoplasm apart from proliferation needs to be further explored.