RNA-binding proteins (RBPs) perform key functions in posttranscriptional regulation, adding complexity to the RNA life cycle. RNA interactome capture techniques have been applied to various organisms of interest and detected hundreds of RBPs, some with uncharacterized functions. However, even in many well-studied organisms, the primary sequence motif for most RBPs remains unknown. Here, we describe a 3-day protocol where users couple an RNA sequence of interest that is known to be bound by an RBP(s) with agarose beads, incubate the now tagged RNA sequence with protein lysate, and then pull down the proteins bound to the RNA. Subsequent mass spectrometry allows users to profile the RNA sequence-interacting proteome and pick out any enriched proteins as RBPs of interest. This protocol allows researchers to match sequences to their RBPs and even often identify novel RBPs or new functions for known RBPs.Double-stranded RNA (dsRNA) plays an essential role in many biological processes and has a great potential for agronomic applications in disease and pest control. A simple and effective method to monitor dsRNA uptake by fungi is crucial for the use of dsRNA as alternative fungicide. The protocol reported in this chapter describes an efficient method to detect and localize labeled dsRNA in fungal hyphae. We use the fungal Verticillium longisporum, a fungal plant pathogen that commonly infects rapeseed and other Brassica species, to explain the procedure, though we have validated the method in a broad spectrum of fungi. Hereafter we elucidate step-by-step the production, fluorescence labeling, as well as detection of dsRNA via fluorescence microscopy in fungal mycelium.Fungal pathogens are responsible for severe crop losses worldwide. Defending crops against fungal disease is critical for global food security; however, most current disease management approaches rely on chemical fungicides that can leave dangerous residues in the environment. RNA interference (RNAi) is an important process through which RNA molecules target and silence complementary genes, regulating gene expression during both transcription and translation. Recently, it has been discovered that some species of fungi can efficiently take up RNAs originating from their host plant and the environment. If these RNAs are complementary to fungal genes, this can lead to the targeting and silencing of fungal genes, termed “cross-kingdom RNAi,” if the RNA originated from a plant host, or “environmental RNAi,” if the RNA originated from the environment. These discoveries have inspired the development of spray-induced gene silencing (SIGS), an innovative crop protection strategy involving the foliar application of RNAs which target and silence fungal virulence genes for plant protection against fungal pathogens. The effectiveness of SIGS is largely dependent on the ability of fungi to take up environmental RNAs. see more Here, we describe the protocols used to label and visualize RNAs which are taken up by Botrytis cinerea. This protocol could easily be adapted for use across various fungal species. Determining the efficiency of RNA uptake by a specific fungal species is a critical first step to determining if SIGS approaches could be an effective control strategy for that fungus.Intercellular communication is a major hallmark of multicellular organisms and is responsible for coordinating cell and tissue differentiation, immune responses, synaptic transmission, and both paracrine and endocrine signaling, for example. Small molecules, peptides, and proteins have all been studied extensively as mediators of intercellular communication; however, RNAs have also been shown recently to transfer between cells. In mammalian cells, microRNAs, tRNAs, short noncoding RNAs, mRNA fragments, as well as full-length mRNAs have all been shown to transfer between cells either by exosomes or by membrane nanotubes. We have previously described nanotube-mediated cell-cell transfer of specific mRNAs between heterologous mammalian cell types cultured in vitro. Here, we describe a simple method for the unbiased and quantitative identification of the complete range of transferred mRNAs (i.e., the mRNA transferome) in one population of mammalian cells following co-culture with another population. After co-culture, the individual cell populations are sorted by magnetic bead-mediated cell sorting and the transferred RNAs are then identified by downstream analysis methods, such as RNA sequencing. Application of this technique not only allows for determination of the mRNA transferome, but can also reveal changes in the native transcriptome of a cell population after co-culture. This can indicate the effect that co-culture and intercellular transfer of mRNA have upon cell physiology.Mobility assays coupled with RNA profiling have revealed the presence of hundreds of full-length non-cell-autonomous messenger RNAs that move through the whole plant via the phloem cell system. Monitoring the movement of these RNA signals can be difficult and time consuming. Here we describe a simple, virus-based system for surveying RNA movement by replacing specific sequences within the viral RNA genome of potato virus X (PVX) that are critical for movement with other sequences that facilitate movement. PVX is a RNA virus dependent on three small proteins that facilitate cell-to-cell transport and a coat protein (CP) required for long-distance spread of PVX. Deletion of the CP blocks movement, whereas replacing the CP with phloem-mobile RNA sequences reinstates mobility. Two experimental models validating this assay system are discussed. One involves the movement of the flowering locus T RNA that regulates floral induction and the second involves movement of StBEL5, a long-distance RNA signal that regulates tuber formation in potato.Subcellular localizations of RNAs can be imaged in vivo with genetically encoded reporters consisting of a sequence-specific RNA-binding protein (RBP) fused to a fluorescent protein. Several such reporter systems have been described based on RBPs that recognize RNA stem-loops. Here we describe RNA tagging for imaging with an inactive mutant of the bacterial endonuclease Csy4, which has a significantly higher affinity for its cognate stem-loop than alternative systems. This property allows for sensitive imaging with only few tandem copies of the target stem-loop inserted into the RNA of interest.