An improved MS2 system for accurate reporting of the mRNA life cycle

Tutucci, Evelina; Vera, María; Biswas, Jeetayu; Garcia, Jennifer F.; Parker, Roy; Singer, Robert H.

doi:10.1038/nmeth.4502

Cited by 262 publications

(312 citation statements)

References 42 publications

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“…1A, (Lionnet et al, 2011b)). mRNA was imaged with smFISH Additionally, the MCP consensus binding sequence was utilized to perform an in-silico search for MS2 binding sites across the transcriptome (Tutucci et al, 2018). All three approaches were successful in predicting MCP binding to the target sites ( Fig.…”

Section: Resultsmentioning

confidence: 99%

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Biswas

Rahman

Gupta³

et al. 2019

Preprint

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Nearly every step of RNA regulation is mediated by binding proteins (RBPs). The most common method to identify specific RBP target transcripts in vivo is by crosslinking ("CLIP" and its variants), which rely on protein-RNA crosslinking and specific antibodies. Another recently introduced method exploits RNA editing, with the catalytic domain of ADAR covalently attached to a specific RBP ("TRIBE"). Both approaches suffer from difficulties in distinguishing real RNA targets from false negative and especially false positive signals. To critically evaluate this problem, we used fibroblasts from a mouse where every endogenous β -actin mRNA molecule was tagged with the bacteriophage MS2 RNA stem loops; hence there is only a single bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE could both detect the single RNA target, albeit with some false positives (transcripts lacking the MS2 stem loops). Consistent false positive CLIP signals could be attributed to nonspecific antibody crosslinking. To our surprise, the supposed false positive TRIBE targets correlated with the location of genes spatially proximal to the β -actin gene. This result indicates that MCP-ADAR bound to β -actin mRNA contacted and edited nearby nascent transcripts, as evidenced by frequent intronic editing. Importantly, nascent transcripts on nearby chromosomes were also edited, agreeing with the interchromosomal contacts observed in chromosome paint and Hi-C. The identification of nascent RNA-RNA contacts imply that RNA-regulatory proteins such as splicing factors can associate with multiple nascent transcripts and thereby form domains of post-transcriptional activity, which increase their local concentrations. These results more generally indicate that TRIBE combined with the MS2 system, MS2-TRIBE, is a new tool to study nuclear RNA organization and regulation.3 Introduction:

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Section: Resultsmentioning

confidence: 99%

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

Biswas

Rahman

Gupta³

et al. 2019

Preprint

Self Cite

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“…Therefore, while this approach is very useful in revealing transcriptional activity, results have to be carefully interpreted when applied to studies of RNA degradation and localization. Recently, a re-engineered MS2 system has been developed that has minimal effect on mRNA half-life (45). In addition, an mRNA degradation assay using rifampicin can provide a good test on potential effects of the labeling method on mRNA turnover.…”

Section: Approaches To Study Rna Localizationmentioning

confidence: 99%

RNA Localization in Bacteria

Fei

Sharma

2018

Microbiol Spectr

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Diverse mechanisms and functions of post-transcriptional regulation by small regulatory RNAs (sRNAs) and RNA binding proteins (RBPs) have been described in bacteria. In contrast, little is known about the spatial organization of RNAs in bacterial cells. In eukaryotes, subcellular localization and transport of RNAs play important roles in diverse physiological processes, such as embryonic patterning, asymmetric cell division, epithelial polarity, and neuronal plasticity. It is now clear that bacterial RNAs also can accumulate at distinct sites in the cell. However, due the small size of bacterial cells, localized RNA and translation are more challenging to study in bacterial cells, and the underlying molecular mechanisms of transcript localization are less understood. Here we review the emerging examples of RNAs localized to specific subcellular locations in bacteria, with indications that subcellular localization of transcripts might be important for regulatory processes. Diverse mechanisms for bacterial RNA localization have been suggested, including close association to their genomic site of transcription, or to the localizations of their protein products in translation-dependent or independent processes. We also provide an overview of the state-of-the-art of technologies to visualize and track bacterial RNAs, ranging from hybridization-based approaches in fixed cells to in vivo imaging approaches using fluorescent protein reporters and/or RNA aptamers in single living bacterial cells. We conclude with a discussion of open questions in the field and ongoing technological developments regarding RNA imaging in eukaryotic systems that might likewise provide novel insights into RNA localization in bacteria.

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“…21,22 However, mRNAs detected by localized tagging might be translated, but in a manner that escapes detection in profiling, which requires ribosomes with their exit tunnels near the membrane 21,22 . Underlying mechanisms may be identified through use of multiple tagging devices in the same cell, live imaging, 8,[18][19][20]90 or strategies that track localization and translation simultaneously 91,92 .…”

Section: Discussionmentioning

confidence: 99%

“…Localization hinges on interplay between sequences in the RNAs, RNA binding proteins, molecular motors, and subcellular structures, such as the ER or cytoskeleton [10][11][12][13] . Advancements in FISH [14][15][16][17] , live imaging [18][19][20] and sequencing-based methods including proximity-specific ribosome profiling 21,22 , APEX-RIP 23 and APEX-Seq 24,25 are very powerful, but typically provide snapshots of localized RNAs, since cells do not survive the required treatments. They also often require either custom oligonucleotide probes or sophisticated equipment.…”

Section: Introductionmentioning

confidence: 99%

Records of RNA localization through covalent tagging

Medina-Munoz

Lapointe

Porter

et al. 2019

Preprint

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RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific subcellular locations, we anchored a uridine-adding enzyme at those sites, which then marked RNAs in its vicinity with 3' terminal uridines. RNAs were tagged independent of their translation status, and included not only mRNAs, but also ncRNAs and ncRNA processing intermediates. A battery of RNAs, including the stress sensor, IRE1, were tagged at both ER and mitochondria, and reveal RNAs whose dual localization is conserved from yeast to human cells.

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An improved MS2 system for accurate reporting of the mRNA life cycle

Cited by 262 publications

References 42 publications

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

MS2-TRIBE evaluates protein-RNA interactions and nuclear organization of transcription by RNA editing

RNA Localization in Bacteria

Records of RNA localization through covalent tagging

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