Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

Engel, Krysta; Lo, Hei‐Yong G.; Goering, Raeann; Liu, Ying; Spitale, Robert C.; Taliaferro, J. Matthew

doi:10.1093/nar/gkab1185

Cited by 29 publications

(41 citation statements)

References 70 publications

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“…We used our recently developed proximity labeling technique Halo-seq (Engel et al, 2021) to assay RNA localization across the apicobasal axis of C2bbe1 monolayers ( Figure 1B ). Halo-seq relies on the expression of a Halo-tagged protein with specific localization to a subcellular compartment of interest.…”

Section: Resultsmentioning

confidence: 99%

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RNA localization mechanisms transcend cell morphology

Goering

Arora

Taliaferro

2022

Preprint

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RNA molecules are localized to specific subcellular regions through interactions between RNA regulatory elements and RNA binding proteins (RBPs). Generally, our knowledge of the mechanistic details behind the localization of a given RNA is restricted to a particular cell type. Here, we show that RNA/RBP interactions that regulate RNA localization in one cell type predictably regulate localization in other cell types with vastly different morphologies. To determine transcriptome-wide RNA spatial distributions across the apicobasal axis of human intestinal epithelial cells, we used our recently developed RNA proximity labeling technique, Halo-seq. We found that mRNAs encoding ribosomal proteins (RP mRNAs) were strongly localized to the basal pole of these cells. Using reporter transcripts and single molecule RNA FISH, we found that pyrimidine-rich TOP motifs in the 5′ UTRs of RP mRNAs were sufficient to drive basal RNA localization. Interestingly, the same TOP motifs were also sufficient to drive RNA localization to the neurites of mouse neuronal cells. In both cell types, the regulatory activity of the TOP motif was dependent on it being at the extreme 5′ end of the transcript, was abolished upon perturbation of the TOP-binding protein LARP1, and was reduced upon inhibition of kinesins. To extend these findings, we compared subcellular RNAseq data from neuronal and epithelial cells. We found that the basal compartment of epithelial cells and the projections of neuronal cells were enriched for highly similar sets of RNAs, indicating that broadly similar mechanisms may be transporting RNAs in both cell types. These findings identify the first RNA element known to regulate RNA localization across the apicobasal axis of epithelial cells, establish LARP1 as an RNA localization regulator, and demonstrate that RNA localization mechanisms cut across cell morphologies.

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Section: Resultsmentioning

confidence: 99%

“…Halo-seq was performed as previously described (Engel et al, 2021). Details regarding each step of Halo-seq are provided below.…”

Section: Halo-seqmentioning

confidence: 99%

RNA localization mechanisms transcend cell morphology

Goering

Arora

Taliaferro

2022

Preprint

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“…Additionally, using machine learning models to predict novel PAS (Li et al, 2021;Lusk et al, 2021) and improvements in quantification methods to analyze different isoforms in spatially restricted transcriptomes will greatly improve our understanding of the APA regulation and how it affects RNA localization. In recent years, proximity-based labeling methods (Fazal et al, 2019;Engel et al, 2021) have enabled the study of RNA localization in diverse cell types. These advancements combined may further increase the number of examples where APA affects RNA localization and thereby allow understanding of generalized mechanisms by which the two are coregulated.…”

Section: Discussionmentioning

confidence: 99%

“…These results bolster the notion that two distinct mechanisms of RNA localization to the ER exist: co-translational localization, and translationindependent, 3′ end mediated localization. However, further experimentation with more precise techniques such as proximity labeling assays, like APEX-seq (Fazal et al, 2019) or HaLo-seq (Engel et al, 2021), would aid in further elucidating two separate mechanisms of RNA localization to the ER.…”

Section: Apa and Rna Localization To Membranesmentioning

confidence: 99%

The Role of Alternative Polyadenylation in the Regulation of Subcellular RNA Localization

Arora

Goering

et al. 2022

Front. Genet.

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Alternative polyadenylation (APA) is a widespread and conserved regulatory mechanism that generates diverse 3′ ends on mRNA. APA patterns are often tissue specific and play an important role in cellular processes such as cell proliferation, differentiation, and response to stress. Many APA sites are found in 3′ UTRs, generating mRNA isoforms with different 3′ UTR contents. These alternate 3′ UTR isoforms can change how the transcript is regulated, affecting its stability and translation. Since the subcellular localization of a transcript is often regulated by 3′ UTR sequences, this implies that APA can also change transcript location. However, this connection between APA and RNA localization has only recently been explored. In this review, we discuss the role of APA in mRNA localization across distinct subcellular compartments. We also discuss current challenges and future advancements that will aid our understanding of how APA affects RNA localization and molecular mechanisms that drive these processes.

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“…In addition, nucleic acid labeling by organic or genetically encoded photosensitizers using propargyl amine (PA) has been developed. 63 − 66 It is expected that PA can be conjugated to the oxidized base products of the nucleic acids by proximal photocatalysis reactions. 64 , 65 …”

Section: Photocatalyst-mediated Plmentioning

confidence: 99%

Molecular Spatiomics by Proximity Labeling

Kang

Rhee

2022

Acc. Chem. Res.

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Conspectus Proximity labeling can be defined as an enzymatic “in-cell” chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not “accessible” via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell–cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in “spatiomics,” that is, for the extraction of spatially distributed biological information in a cell or organism. Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This “in-cell” reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models. Simultaneously, chemists have also adopted the concept and employed chemical “photocatalysts” as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.

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Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

Cited by 29 publications

References 70 publications

RNA localization mechanisms transcend cell morphology

RNA localization mechanisms transcend cell morphology

The Role of Alternative Polyadenylation in the Regulation of Subcellular RNA Localization

Molecular Spatiomics by Proximity Labeling

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