Records of RNA localization through covalent tagging

Medina-Munoz, Hugo C; Lapointe, Christopher P.; Porter, Douglas F.; Wickens, Marvin

doi:10.1101/785816

Cited by 3 publications

(6 citation statements)

References 102 publications

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“…By modifying the sequences in the linker regions of our reporter (currently 50bp RNA linker flanking MS2 stem-loops), we can evaluate enzymes with distinct sequence context preferences, paving the way for engineered rBEs, as has been previously conducted with DNA editors 8,26,27 . Additionally, there is vast potential to evaluate the ability of rBEs to detect localized RNAs 49,50 . For example, integrating the molecular recording strategy, localized RNA recording, and proximity-specific ribosome profiling data from yeast added insights into the interplay between RNA localization and translation at the ER and mitochondria that is impossible with either method alone 49–52 .…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, there is vast potential to evaluate the ability of rBEs to detect localized RNAs 49,50 . For example, integrating the molecular recording strategy, localized RNA recording, and proximity-specific ribosome profiling data from yeast added insights into the interplay between RNA localization and translation at the ER and mitochondria that is impossible with either method alone 49–52 . Adapting optimized rBEs to identify localized RNAs can expand on the localized RNA recording strategy and complement methods like APEX-Seq and proximity- specific ribosome profiling 49–53 .…”

Section: Discussionmentioning

confidence: 99%

“…For example, integrating the molecular recording strategy, localized RNA recording, and proximity-specific ribosome profiling data from yeast added insights into the interplay between RNA localization and translation at the ER and mitochondria that is impossible with either method alone 49–52 . Adapting optimized rBEs to identify localized RNAs can expand on the localized RNA recording strategy and complement methods like APEX-Seq and proximity- specific ribosome profiling 49–53 . Here, distinct editors may also be used to record RNA molecules that interact with multiple sub-cellular locales, as previously suggested with combined localized RNA recording and TRIBE 49,50 .…”

Section: Discussionmentioning

confidence: 99%

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Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies

Medina-Munoz,

Kofman,

Jagannatha

et al. 2023

Preprint

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RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNAinteraction-based triaging ofenzymes that editRNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies

Medina-Munoz,

Kofman,

Jagannatha

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…In addition, the integration of RNA editing signal over time is an advantage compared with techniques requiring cellular fixation. Expression of RBP fusions (such as in MS2-TRIBE and also in other recently reported methods, Medina-Muñoz et al., 2019 ) may allow for RBPs to bind to a larger proportion of their targets when integrated over time, leading to more efficient identification of targets.…”

Section: Discussionmentioning

confidence: 99%

MS2-TRIBE Evaluates Both Protein-RNA Interactions and Nuclear Organization of Transcription by RNA Editing

et al. 2020

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Summary Both UV-cross-linking and immunoprecipitation (CLIP) and RNA editing (TRIBE) can identify the targets of RNA-binding proteins. To evaluate false-positives of CLIP and TRIBE, endogenous β-actin mRNA was tagged with MS2 stem loops, making it the only bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE detected β-actin, albeit with false-positives. False-positive CLIP signals were attributed to nonspecific antibody interactions. In contrast, putative false-positive TRIBE targets were genes spatially proximal to the β-actin gene. MCP-ADAR edited nearby nascent transcripts consistent with interchromosomal contacts observed in Hi-C. The identification of nascent contacts implies RNA regulatory proteins (e.g., splicing factors) associated with multiple nascent transcripts, forming domains of post-transcriptional activity. Repeating these results with an integrated inducible MS2 reporter indicated that MS2-TRIBE can be applied to a broad array of cells and transcripts to study spatial organization and nuclear RNA regulation.

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“…One such method is based on spatially restricted nucleobase oxidation, which uses localized fluorophores ( Li et al 2017 ). Another approach uses an enzyme to add uridine residues to RNAs in specific locations in Caenorhabditis elegans , including in the mitochondria and ER ( Medina-Muñoz et al 2019 ). A third approach, called CAP-seq, uses light-activated, proximity-dependent photo-oxidation of RNA ( Wang et al 2019b ).…”

Section: Modern Approaches To Study Rna Localizationmentioning

confidence: 99%

Subcellular Spatial Transcriptomes: Emerging Frontier for Understanding Gene Regulation

Fazal

Chang

2019

Cold Spring Harb Symp Quant Biol

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RNAs are trafficked and localized with exquisite precision inside the cell. Studies of candidate messenger RNAs have shown the vital importance of RNA subcellular location in development and cellular function. New sequencing-and imaging-based methods are providing complementary insights into subcellular localization of RNAs transcriptome-wide. APEX-seq and ribosome profiling as well as proximity-labeling approaches have revealed thousands of transcript isoforms are localized to distinct cytotopic locations, including locations that defy biochemical fractionation and hence were missed by prior studies. Sequences in the 3′ and 5′ untranslated regions (UTRs) serve as "zip codes" to direct transcripts to particular locales, and it is clear that intronic and retrotransposable sequences within transcripts have been co-opted by cells to control localization. Molecular motors, nuclear-to-cytosol RNA export, liquid-liquid phase separation, RNA modifications, and RNA structure dynamically shape the subcellular transcriptome. Location-based RNA regulation continues to pose new mysteries for the field, yet promises to reveal insights into fundamental cell biology and disease mechanisms.

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Records of RNA localization through covalent tagging

Cited by 3 publications

References 102 publications

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies

MS2-TRIBE Evaluates Both Protein-RNA Interactions and Nuclear Organization of Transcription by RNA Editing

Subcellular Spatial Transcriptomes: Emerging Frontier for Understanding Gene Regulation

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