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Published in final edited form as: Biochim Biophys Acta Mol Cell Res. 2022 Jan 6;1869(4):119202. doi: 10.1016/j.bbamcr.2021.119202

TRANSCRIPTOME-SCALE METHODS FOR UNCOVERING SUBCELLULAR RNA LOCALIZATION MECHANISMS

J Matthew Taliaferro 1
PMCID: PMC9035289  NIHMSID: NIHMS1799419  PMID: 34998919

Abstract

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.

Keywords: RNA localization, RNA transport, Cell fractionation, Proximity labeling, MPRA

1. INTRODUCTION

A hallmark of essentially all cells is the non-uniform distribution of proteins within them. This asymmetric patterning of proteins contributes to the definition of spatially defined regions with specialized activities. In cells with complex morphologies like neurons, the asymmetry can be extreme with cellular compartments at extraordinary distances from each other. Although this asymmetry can be achieved through the targeting of proteins to desired locations [1], it is often also accomplished through the targeting of the instructions for making the protein (i.e. the RNA) [2]. This strategy allows the protein to be translated on-site, immediately producing a correctly localized protein and preventing the deleterious effects of a mislocalized protein.

Misregulation of RNA localization is associated with a range of cellular and organismal phenotypes. In Drosophila, specific RNAs localized to the anterior and posterior poles define the anterior/posterior axis of the developing embryo [3-5], and mislocalization of these transcripts results in developmental patterning defects [5-7]. In mammals, particularly in neurons, RNA localization and localized translation are important for cell function [8]. Mutations in RNA binding proteins (RBPs) that regulate RNA localization are associated with neurological diseases, including Fragile X syndrome, Spinal Muscular Atrophy, and Amyotrophic Lateral Sclerosis [9-12].

Localized RNAs often contain sequences that regulate the trafficking of the molecule by serving as binding sites for RBPs that mediate the transport [13]. These sequences, often called “zipcodes”, are usually found in the 3′ UTRs of localized transcripts [2]. The majority of the currently known zipcodes are relatively long (> 50 nt) [13,14], making them much larger than regulatory elements that control other processes like RNA stability and splicing, which tend to be 4 to 8 nucleotides long [15].

In addition to RBP-based mechanisms of RNA transport, RNAs can also become asymmetrically distributed through other processes, including localized changes in transcript stability [16,17], diffusion and entrapment [18], and co-translational transport of nascent peptides [19]. All of these mechanisms at some level depend on specific sequences contained within the localized transcript.

The increased size of RNA localization regulatory elements implies that they may be largely defined by their secondary structure. It also makes their identification through computational methods very difficult. Perhaps unsurprisingly then, although thousands of RNAs have been found to be specifically localized in a variety of contexts [20-22], the identity of the zipcode/RBP cognate pairs that regulate transport are known for only a handful of these [13].

In the following sections, I will describe experimental approaches that have been used to interrogate these mechanisms as well as newer techniques that rely on high-throughput sequencing to assess the localization of thousands of transcripts at once.

2. CLASSICAL APPROACHES FOR THE STUDY OF RNA LOCALIZATION MECHANISMS

Historically, investigations into mechanisms regulating RNA localization have generally been undertaken for one localized transcript at a time. These approaches involve systematically testing the ability of RNA sequences derived from the localized transcript to induce localization of an unrelated reporter transcript (Fig. 1). The entire 3′ UTR of the localized transcript is commonly used as a starting point for these experiments [23-26]. If the 3′ UTR is sufficient to direct localization of the reporter transcript, then progressively smaller pieces of the 3′ UTR can be tested until a minimal element is identified [24,27,28]. These minimal elements can then serve as useful handles for further exploration of the requirements for localization, including secondary structure and the identity of RBPs that bind the sequence [29,30].

Figure 1.

Figure 1.

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Classical strategies for identifying RNA localization regulatory elements. Once a localized RNA is identified, pieces of the RNA are fused to a reporter transcript, and the localization of the resulting reporter is assayed. If the fused transcript pieces confers localization activity to the reporter, then the regulatory element can be identified with greater resolution by iteratively decreasing the length of the tested sequence and assaying its localization-inducing activity.

Over the past few decades, these experiments have formed the basis of our knowledge of the regulation of RNA localization. However, they are very laborious and are limited in their throughput by their reliance on the optical assessment of individual reporters. Further, in the end, what they reveal are the requirements for localization for the single transcript that was tested. It can be difficult to generalize results from these single transcript studies into more global rules that regulate RNA localization.

3. HIGH-THROUGHPUT SEQUENCING-BASED TRANSCRIPTOMIC METHODS FOR IDENTIFYING LOCALIZED RNAS

It is unlikely that each localized RNA uses a localization mechanism that is unique to itself. Instead, mechanisms are likely shared across groups of localized transcripts. Therefore, by looking at the members of such groups, it might be possible to derive the elements regulating their transport by looking for common features shared among them. This is particularly true when analyzing changes in localized RNAs in response to the perturbation of specific localization mechanisms.

Recently developed techniques use high-throughput sequencing to identify sets of transcripts enriched at particular subcellular locations. Once this set of localized transcripts has been defined, potential localization regulatory elements can be identified by looking for sequences enriched in localized RNAs compared to nonlocalized RNAs. Although these elements usually require validation using the more classical techniques described above, multiple studies using transcriptome-scale approaches have increased our knowledge of general RNA features associated with RNA localization [12,21,22,31-33].

At their core, each of these studies relied on the isolation and analysis of subcellular transcriptomes. Techniques for generating these transcriptomes are rapidly evolving but can be currently classified into one of the seven main themes outlined below. In each section, the relative strengths and limitations of each approach will be discussed.

3.1. CELLULAR FRACTIONATION USING TOMOGRAPHY

If the system being studied is large enough, then the cell or tissue can be systematically sliced into a series of fractions using tomography. RNA can then be isolated from each of these fractions and analyzed, and the relative localization of each transcript within the transcript can then be reconstructed.

Generally, this technique does not have subcellular resolution and has instead been applied to embryos or tissues, including zebrafish and Drosophila embryos [34,35] and zebrafish hearts [36]. Although the results from these techniques have not yet been used to define RNA features associated with the observed localization patterns, tomography approaches hold the promise of allowing precise control over the spatiotemporal parameters of fractionation.

3.2. IN SITU SEQUENCING USING PATTERNED ARRAYS

The use of patterned DNA arrays for the collection of spatially defined high-throughput sequencing data has been successfully applied to single cell RNAseq experiments, allowing the reconstruction of tissue-level RNA expression patterns [37,38]. In these techniques, tissues are applied to substrates that contain patterned DNA barcodes. These barcodes are incorporated into RNAseq libraries that are prepared in situ. The locations of these barcodes can then be traced back to their positions on the substrate, allowing the intersection of RNA expression and spatial information. Until recently, the resolution afforded by these techniques (i.e. the space between adjacent DNA barcodes) generally restricted these techniques to tissue-level analyses.

However, advances have increased this resolution from the range of tens of microns to hundreds of nanometers. In the newly developed Stereo-seq, DNA nanoballs are patterned onto a substrate chip [39]. As before, barcodes contained within these nanoballs are incorporated into RNAseq libraries that are created in situ. The reduced distance between adjacent barcodes allows up to 400 unique barcodes to be contained within the size of a typical mammalian cell. This approach has been used to profile expression patterns within the developing Drosophila embryo [40]. Although the syncytial Drosophila is much larger than most mammalian cells, this provides an exciting proof of concept of the technique and its application to subcellular RNA expression patterns.

3.3. CELLULAR FRACTIONATION USING MICROPOROUS MEMBRANES

Cells with long extensions, like neurons, can be mechanically fractionated into cell body and projection fractions. RNA can then be collected from both of these fractions and analyzed using high-throughput sequencing. Localized RNAs are identified as those significantly more abundant in one fraction than the other.

Cells can be grown on membranes that contain pores, often 1 to 3 microns in diameter [22,32,41] (Fig. 2A). These pores are large enough for projections to grow down through the pores to the underside of the membrane yet are small enough such that cell bodies are restricted to the top. By scraping the top of the membrane, cells can then be mechanically fractionated into cell body and projection samples, and RNA can be isolated from each sample. Although this technique is widely used with neuronal cells, it can be used to monitor RNA localization to other cellular projections, including those of fibroblasts and migrating cancer cells [32,42].

Figure 2.

Figure 2.

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Cellular fractionation methods for subcellular transcriptomic analysis. Following RNA isolation from subcellular compartments, the RNA can be profiled using high-throughput sequencing. (A) Cells that contain long, thin projections can be grown on microporous membranes in which the projections grow down through the membrane. Following growth, cells can be mechanically fractionated into cell body and projection fractions. (B) Alternatively, neuronal cells can be grown in microfluidic culture systems in which cell bodies and projections are maintained in hydrostatically separated vessels. Following cell lysis, the separation between compartments is maintained with hydrostatic pressure. (C) Laser capture microdissection can be used to isolate specific subcellular locations. Following their isolation, RNA is purified from these microdissected regions and prepared for analysis. (D) A variety of cell types can be separated into biochemically defined fractions using combinations of detergents and differential centrifugation. (E) Proximity labeling approaches use the activity of specifically localized proteins to label nearby RNAs, facilitating their purification and quantification.

3.4. CELLULAR FRACTIONATION USING MICROFLUIDIC CULTURE SYSTEMS

Growth on porous membranes can effectively separate cell bodies and projections, but it does not give much control over the length of the projection to be interrogated. For example, axonal and dendritic projections can grow through the pores, making it impossible to distinguish their RNA contents using this method.

To give greater spatial resolution during cell fractionation, microfluidic culture systems have been developed in which cell body and projection compartments are hydrostatically separated [43] (Fig. 2B). The distance between the compartments can be changed, allowing a more precise isolation of projections that are at least a given length away from cell bodies. These culture systems have been used to identify localized RNAs in mouse and human neurons [44-46] as well as for monitoring the translation of localized RNAs [47].

3.5. CELLULAR FRACTIONATION USING LASER CAPTURE MICRODISSECTION

If the cellular region of interest is easily identifiable using microscopy, then the region can be directly dissected out of the cell or tissue sample using laser capture microdissection (Fig. 2C). With this approach, regions of interest are first located visually and then separated from the rest of the sample through the use of a targeted laser [48]. The action of the laser facilitates collection of the sample, after which RNA can be collected and analyzed.

This technique has been used to interrogate the RNA content of neuronal subcompartments [49-51] as well as the apical and basolateral compartments of intestinal epithelial cells [52]. Although these techniques give precise control over the subcellular location to be interrogated, the RNA amounts recovered from the microdissected sections are quite small, necessitating the use of specialized library preparation and next-generation sequencing protocols.

3.6. CELLULAR FRACTIONATION USING BIOCHEMICAL METHODS

The techniques described above rely either on a particular cellular morphology that is most commonly found in neurons or in large systems amenable to tomography. However, RNAs are specifically localized in a wide range of cell types, many of which do not form long projections or are large enough for tomography. To study RNA localization in these cell types, biochemical techniques have been applied in which cells are fractionated using detergent-containing buffers and differential centrifugation (Fig. 2D). As with the mechanical fractionation methods, RNA is isolated from each fraction and then analyzed using high-throughput sequencing.

These techniques have been used to investigate the RNA contents of biochemically defined cellular fractions [53,54] as well as define transcript features associated with localization to each fraction. They have also been used to study RNAs trafficked to cytoskeletal substructures [55,56] and organelles [57].

3.7. ISOLATION OF LOCALIZED RNAS USING PROXIMITY LABELING

The use of biochemical fractionation techniques for the study of RNA localization has removed the reliance of cell fractionation on cell morphology or size. However, they have their weaknesses. Most notably, the copurification of two RNA molecules into a biochemically defined fraction is no guarantee that they were spatially coincident within the intact cell. Transcripts that were spatially separated within the cell but contained within environments with similar biochemical properties may copurify, leading to false positives. Conversely, the relationship between spatially proximal RNAs may not survive biochemical treatments, leading to false negatives.

As a counter to these issues, a handful of RNA proximity labeling techniques have been developed that specifically label spatially defined RNA populations. These labels can then be used as handles with which to purify the transcripts and facilitate their analysis. Importantly, with these techniques, the spatially sensitive RNA labeling steps are performed while the cell is alive and intact, obviating concerns about spurious copurification.

Generally, these methods rely on a protein marker that is specifically localized to the subcellular region of interest (Fig. 2E). Reactive chemical species are produced in this location, labeling RNAs in the vicinity, with a resolution of approximately 20 nm. In the APEX-seq technique [58,59], these reactive species are produced by the enzyme APEX2. Radical generation is spatially restricted by genetically fusing APEX2 to a localized protein. When a nearby RNA molecule is attacked by APEX-generated radicals, it can then be further attacked by biotin phenol, resulting in its biotinylation. Localized RNAs can subsequently be purified from the bulk RNA sample using streptavidin and profiled using high-throughput sequencing.

CAP-seq uses a similar approach [60]. Radicals are generated in an optically controlled manner by the enzyme miniSOG2. As with APEX-seq, the subcellular localization of miniSOG2 can be controlled through genetic fusion to specific marker proteins. miniSOG2-proximal labeled RNAs are substrates for nucleophilic attack by propargylamine, resulting in their alkynylation. After isolation of total cellular RNA, alkynylated RNAs can be biotinylated in vitro through the use of Cu(I)-catalyzed azide-alkyne cycloaddition (“Click”) chemistry.

A third RNA proximity labeling technique, Halo-seq, was recently reported [61]. In this approach, reactive species are produced by the small molecule dibromofluorescein (DBF) following its irradiation with green light. The subcellular location of DBF is controlled through its incorporation into a Halo ligand. Halo ligands are a class of small molecules that selectively and covalently attach themselves to HaloTag domains [62]. By genetically fusing a HaloTag domain to a spatially restricted protein, the localization of DBF is also restricted. As with CAP-seq, DBF-proximal labeled RNAs are alkynylated, making them substrates for in vitro biotinylation.

These proximity labeling techniques have been used to identify localized RNA populations at a wide variety of subcellular locations [58-61]. The flexibility they possess in terms of the subcellular locations available for study make them exciting technical additions.

4. HIGH-THROUGHPUT APPROACHES FOR DIRECTLY IDENTIFYING REGULATORY ELEMENTS

The above techniques for isolating subcellular transcriptomes are informative for understanding which RNAs are at a given location, but they do not directly give information on how they got there. By combining subcellular transcriptomics with RBP perturbations and massively parallel reporter assays (MPRAs), a richer mechanistic view of the sequence and RBP requirements for RNA transport can be obtained.

4.1. PROFILING RNA LOCALIZATION FOLLOWING RBP PERTURBATION

As discussed above, knowing the identities of many RNAs enriched at a particular location can allow the discovery of sequence requirements that regulate the localization of groups of transcripts through the discovery of patterns shared among them. However, for many subcellular locations, it is unlikely that all the transcripts enriched at a given site belong to a single such group and are localized there using a single mechanism. A simple analysis, then, of looking for enriched motifs among localized transcripts is often messy because it contains the summed signal from multiple RNA localization mechanisms.

In order to zero in on RNA localization regulatory elements, it can be helpful to focus on one RNA localization mechanism. One way this can be done is through the perturbation of specific RBPs thought to be involved in the process. By identifying RNAs that are mislocalized following RBP perturbation, the transcripts that depend upon the RBP for proper localization can be elucidated. If the RBP is directly involved in RNA localization, these RNA localization targets of the RBP should be enriched for its binding sites. Enriched RNA elements can be determined by comparing the sequences of RNAs whose localization was RBP-sensitive to the sequences of RNAs whose localization was RBP-insensitive. Because binding of the RBP to a transcript results in its localization, these binding sites are very often RNA localization regulatory elements. This strategy has been employed with a handful of RBPs, and results from these studies indicate that the localization of dozens of transcripts can be controlled by a single RBP [12,22,32,63,64].

4.2. MASSIVELY PARALLEL REPORTER ASSAYS FOR THE STUDY OF RNA LOCALIZATION

Massively parallel reporter assays (MPRAs) have been successful in identifying regulatory elements that control RNA stability and translation [65,66]. Generally, these experiments test the ability of thousands of RNA sequences to modulate the metabolism of a reporter transcript. These sequences are integrated into a reporter construct in order to create a library of thousands of reporter transcripts, each of which is identical with the exception of the identity of the integrated sequence (Fig. 3). Any differences among the members of this pool of nearly-identical transcripts can therefore be attributed to the variable integrated sequence.

Figure 3.

Figure 3.

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MPRA design and execution. Oligonucleotide sequences drawn from the 3′ UTRs of transcripts are synthesized and incorporated into a reporter transcript. This creates a library of nearly identical reporters whose only differences are in the identity of the incorporated oligonucleotide. This pool of reporters is then expressed in cells, and localized RNA is isolated. Through targeted RNAseq, the abundance of reporter-embedded oligonucleotide sequences in localized and nonlocalized RNA can be quantified.

When MPRAs are applied to RNA localization, they offer the promise of the direct identification of sequences responsible for RNA transport. Three recently reported studies [67-69] demonstrated their use in neuronal systems. In these reports, sequence fragments drawn from naturally occurring 3′ UTRs were integrated into a reporter construct to create a library of reporter transcripts. This library was then expressed in neuronal cells, and the cells were fractionated using microporous membranes. Using targeted RNAseq, the abundance of each integrated sequence in soma and neurite RNA samples was calculated. Sequences that were relatively more abundant in neurite than soma were identified as putative regulators of RNA localization.

Importantly, because the sequence fragments tested in these experiments are relatively small (150-260 nt), the discovery of fragments with RNA localization activity allows immediate identification of active elements (i.e. “zipcodes”) with moderate resolution. Results from MPRA experiments can then be further extended, often with mass spectrometry-based approaches, to identify RBPs that bind identified elements and mediate their transport.

5. CONCLUSION

It should be noted that while the focus of this minireview was on RNA localization techniques that utilize high-throughput sequencing, great advances have also been made in techniques that rely on imaging-based approaches. While the original hybridization-based methods that were developed could only assay a single transcript species at a time, newer techniques have expanded on this paradigm in order to interrogate thousands of transcripts in a single experiment [20,70,71]. When these approaches are combined with expansion microscopy, the resolution afforded by these techniques can be greatly increased [72]. Generally, imaging-based and sequencing-based techniques can be highly complementary, and their concurrent growth may allow for newer experimental designs that further push technical boundaries.

The power of high-throughput techniques to identify components of RNA transport mechanisms lies in their ability to profile hundreds or thousands of transcripts at once. This then gives the opportunity to look for patterns that are enriched in localized RNAs and likely regulate RNA transport. Multiple recently developed techniques for isolating subcellular transcriptomes allow the extension of these high-throughput techniques into previously intractable cellular systems. These approaches complement tried-and-true molecular genetic experiments and represent a promising future for the study of RNA localization.

Inevitably, though, insights gained through transcriptome-scale approaches will need to be validated through more targeted experiments. These experiments often involve the use of reporter transcripts to test the activity of sequence elements identified from high-throughput approaches. In some sense, this places the researcher back at the laborious step of testing the localization of individual sequences (Fig 1). Again, historically, these have been the purview of imaging-based techniques that generally require the interrogation of single transcript species.

However, even here, the power of simultaneously testing the localization of multiple sequences at once holds the promise of reducing experimental workload. In this case, a parallel reporter assay need not be massive, only moderate, to be worthwhile. For example, the ability to simultaneously test the activity of putative localization regulatory elements as well as an array of mutants both immediately provides mechanistic hints and cuts a string of experiments that may take many months into one single undertaking.

FUNDING

This work was funded by the National Institutes of Health (R35-GM133885), the Keck Foundation, and the RNA Bioscience Initiative at the University of Colorado Anschutz Medical Campus. Portions of figures were created with Biorender.com.

Footnotes

DECLARATION OF COMPETING INTEREST

The author declares that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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