Atlas of Subcellular RNA Localization Revealed by APEX-Seq

Fazal, Furqan M.; Han, Shuo; Parker, Kevin R.; Kaewsapsak, Pornchai; Xu, Jin; Boettiger, Alistair N.; Chang, Howard Y.; Ting, Alice Y.

doi:10.1016/j.cell.2019.05.027

Cited by 452 publications

(495 citation statements)

References 125 publications

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“…Although the retained introns are predicted to undergo NMD, we found the retained intron transcripts to be NMD-insensitive. Instead we find they could be detained in the nucleus (Boutz, Bhutkar, and Sharp 2015) , a phenomenon also observed in a recent study on RNA localisation within different cellular compartments (Fazal et al 2018) ( Figure 8 ).…”

Section: The Comparisons Between the Joint Analysis Of Fus Mutationssupporting

confidence: 86%

FUSALS-causative mutations impactFUSautoregulation and the processing of RNA-binding proteins through intron retention

Humphrey

Birsa

Milioto

et al. 2019

Preprint

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Mutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease in which the loss of motor neurons induces progressive weakness and death from respiratory failure, typically only 3-5 years after onset.FUS has been established to have a role in numerous aspects of RNA processing, including splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterised, as most disease models have been based on FUS overexpression, which in itself alters its RNA processing functions. To overcome this, we and others have recently created knock-in models, and have generated high depth RNA-sequencing data on FUS mutants in parallel to FUS knockout. We combined three independent datasets with a joint modelling approach, allowing us to compare the mutation-induced changes to genuine loss of function. We find that FUS ALS-mutations induce a widespread loss of function on expression and splicing, with a preferential effect on RNA binding proteins. Mutant FUS induces intron retention changes through RNA binding, and we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS regulation has been linked to disease, and intriguingly, we find FUS autoregulation to be altered not only by FUS mutations, but also in other genetic forms of ALS, including those caused by TDP-43,

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Section: The Comparisons Between the Joint Analysis Of Fus Mutationssupporting

confidence: 86%

FUSALS-causative mutations impactFUSautoregulation and the processing of RNA-binding proteins through intron retention

Humphrey

Birsa

Milioto

et al. 2019

Preprint

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“…Additionally, we observed no detectable diffusion of biotinylated material outside of the nucleus in our live-cell APEX label and chase experiments ( Figure S1D), which further support the idea that the APEX labeling captured the NL-associated RNAs. We found that previously identified mRNA transcripts with retained introns in the nuclear and APEX-RIP samples which are then mostly, but not completely removed in the cytosolic RNA-seq fraction ( Figure S2D, black bars) (Boutz et al, 2015;Fazal et al, 2019;Lareau et al, 2007).…”

Section: Apex2-lamin-b1 Labeling Identifies Nl-associated Rna With Lomentioning

confidence: 80%

“…Previous studies have used the APEX procedure to isolate RNAs (APEX-RIP) associated with cellular organelles and structures. This APEX-RIP procedure can be done by precipitating protein/RNA complexes, or by directly precipitating RNA since the APEX reaction will label RNA (Fazal et al, 2019;Kaewsapsak et al, 2017). In our experiments, we chose to precipitate protein/RNA complexes in order to isolate NLenriched RNAs since this is expected to yield a more extensive dataset.…”

Section: Apex2-lamin-b1 Labeling Identifies Nl-associated Rna With Lomentioning

confidence: 99%

“…An alternative enzyme suitable for the study of the NL, called Ascorbate Peroxidase (APEX), was recently developed by the Ting group for the purposes of proteomic and RNA identification (Fazal et al, 2019;Hung et al, 2014;Kaewsapsak et al, 2017). This enzyme, which has been extensively engineered into the highly-reactive form, APEX2 (Lam et al, 2015), uses hydrogen peroxide to catalyze the covalent addition of a radicalized biotin-phenol moiety to both protein and RNA species.…”

Section: Introductionmentioning

confidence: 99%

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The versatility of Ascorbate Peroxidase-aided mapping uncovers insights of the nuclear lamina interactions and function

Tran

Paulson

Moresco

et al. 2020

Preprint

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The nuclear lamina (NL) is a proteinaceous network found beneath the inner nuclear membrane. The NL is linked to a number of dynamic cellular activities including chromatin organization, transcription and RNA/protein trafficking through nuclear pores. Our understanding of the NL has been hindered in part by the general insolubility and low extractability of proteins from this region. This has spurred the development of proximity ligation methods that label proteins and/or DNA near the NL for systematic identification (Bar et al., 2018; Chen et al., 2018b; Guelen et al., 2008; Roux et al., 2012). To simplify labeling and improve temporal resolution, we fused APEX2 (Hung et al., 2014; Lam et al., 2015) to the nuclear lamina protein lamin-B1 to map proteins, RNA and DNA associated with the NL. We show that APEX2 labeling of the NL is robust and requires as little as 20 seconds. In addition to identifying the NL proteome, this method revealed NL-proximal RNA species that were largely spliced. These NL-proximal RNAs show a bias toward long 3’ UTRs, suggesting an RNA-regulatory role of the NL. This is further supported by the finding of a bias toward longer 3’ UTRs in genes deregulated in lamin-null cells. Interestingly, these RNAs share a sequence motif in their 3’ UTRs. Finally, we demonstrate that the APEX2 method can reliably map lamina-associated domains (LADs) at different stages of the cell cycle, revealing a variability of short LADs regions enriched for histone lysine 27 trimethylation (H3K27me3). Thus the APEX2 method report here is a useful addition to the molecular toolbox for the study of the NL and permits the identification of proteome, transcriptome, and genome elements associated with this nuclear substructure.

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“…In the 1970s, electron microscopy analysis found that cytoplasmic ribosomes can be localized along the mitochondrial outer membrane (Kellems et al, 1974). Recent microarray and RNA-seq analyses of biochemically fractionated mitochondrial membranes and fluorescent microscopy analysis have identified subsets of nuclear-encoded mRNAs that are mitochondrially localized (Fazal et al, 2019;Gadir et al, 2011;Garcia et al, 2007;Marc et al, 2002;Saint-Georges et al, 2008;Williams et al, 2014). It has been shown that both the 3′ UTR and coding regions, primarily through mitochondrial targeting sequences (MTSs), contribute to mitochondrial localization.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial volume fraction controls translation of nuclear-encoded mitochondrial proteins

Tsuboi

Viana

et al. 2019

Preprint

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Mitochondria are dynamic in their size and morphology yet must also precisely control their protein composition according to cellular energy demand. Although nuclear-encoded mRNAs can be localized to the mitochondrial outer membrane, the importance of this localization in altering mitochondrial protein composition is unclear. We have found that, as yeast switch from fermentative to respiratory metabolism, there is an increase in the fraction of the cytoplasm that is mitochondrial. This drives the localization of certain nuclear-encoded mitochondrial mRNAs to the surface of the mitochondria. Through tethering experiments, we show that mitochondrial mRNA localization is necessary and sufficient to increase protein production to levels required during respiratory growth. Furthermore, we find that ribosome stalling impacts mRNA sensitivity to mitochondrial volume fraction and counterintuitively leads to enhanced protein synthesis by increasing mRNA localization to the mitochondria. This points to a mechanism by which cells are able to use translation elongation and the geometric constraints of the cell to fine-tune organelle-specific gene expression through mRNA localization while potentially circumventing the need to directly coordinate with the nuclear genome. mitochondria | geometric constraints | mRNA localization | translation control

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Atlas of Subcellular RNA Localization Revealed by APEX-Seq

Cited by 452 publications

References 125 publications

FUSALS-causative mutations impactFUSautoregulation and the processing of RNA-binding proteins through intron retention

FUSALS-causative mutations impactFUSautoregulation and the processing of RNA-binding proteins through intron retention

The versatility of Ascorbate Peroxidase-aided mapping uncovers insights of the nuclear lamina interactions and function

Mitochondrial volume fraction controls translation of nuclear-encoded mitochondrial proteins

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