Atlas of Subcellular RNA Localization Revealed by APEX-seq

Abstract

We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.(ERM), which we previously attempted but failed to map using APEX2-ERM and APEX-RIP (Kaewsapsak et al., 2017). When we performed a comparison of APEX-seq and APEX-RIP using HEK 239T cells expressing APEX on the ER membrane (facing the cytosol), we found that APEX-seq could clearly enrich secretory mRNAs on the ER membrane over cytosolic mRNAs, whereas APEX-RIP could not ( Figure 1D). Given the very close proximity between ER membrane-associated RNAs and cytosolic RNAs that are immediately adjacent to the ER, these results suggest that APEX-seq has a labeling radius of only a few nanometers. Validation of APEX-seqEncouraged by the results above, we expanded APEX-seq to nine distinct subcellular locations ( Figure 1E). Correct localization of each APEX2 fusion protein (domain structures shown in Figure S2A) in HEK-293T cells was confirmed by imaging with organelle markers ( Figure 1F). We prepared two replicates for each location ( Figure S2B and S2C, correlation coefficient r between replicates ranged from 0.97 to 1) as well as negative controls in which H2O2 was omitted from the labeling reaction. After labeling for 1 minute and cell lysis, we enriched biotinylated RNAs and prepared APEX-seq libraries with polyA+ selection. Transcripts shorter than 100 nt were excluded during purification. We used DESeq2 (Love et al., 2014) for data analysis and used a qvalue (FDR-adjusted p-value) < 0.05 and a log2fold-change > 0.75 to select for significantly enriched transcripts.Consistent with the RT-qPCR results for the mitochondrial matrix shown in Figure 1C, our sequencing-based analysis showed that all 13 mRNAs and 2 rRNAs encoded by mtDNA are strongly enriched by MITO-APEX2 labeling (Figure 2A, S2D and S2E, mean enrichment > 11fold), whereas no mRNAs encoded by the nuclear genome are enriched. Figure 2B shows good correlation (r > 0.9) between technical replicates.To assess APEX-seq in an "open" subcellular region (not enclosed by a membrane), we focused again on the ER membrane (ERM). The ERM is particularly valuable for methodology validation because there are well-established "true positives" (mRNAs encoding secreted proteins that are known to be translated at the ERM) and well-established "false positive...

Mechanisms and consequences of subcellular RNA localization across diverse cell types

“…In many cell types, mRNA colocalizes with its encoded protein. This has been observed in neurons, 51 Drosophila embryos 52 and HEK293 cells 7 . This phenomenon is suggestive of local translation which has been shown across species and cell types.…”

“…Several neurological diseases, including amyotrophic lateral sclerosis (ALS) and fragile X syndrome (FXS), have been associated with misregulated RNA localization in neurons. Recent advances in high‐throughput sequencing and microscopy techniques have also revealed that RNAs are localized to subcellular compartments (eg, endoplasmic reticulum and mitochondria) in many nonpolarized cell types 6–9 . This observation has led to some key questions.…”

Records of RNA localization through covalent tagging

“…Localization of specific RNAs to the proximity of both ER and the outer mitochondrial membrane is conserved. We compared our tagging results to data recently reported from human embryonic kidney (HEK293T) cells using APEX-Seq 25 . The high fraction of RNAs tagged at both locations in yeast (ER: 268 of 1,148; Mitochondria: 268 of 598, Figure 5A, B) was mirrored in HEK293T cells 25 , as was the identity of many of the RNAs (ERM: 50%; OMM: 67%, Supplementary Fig.…”

“…The classes of Puf3p mRNA targets were retrieved from published biochemical experiments 48 . 25 as used for these analyses. Similar cutoffs applied to our data (log 2 (Fold Change) ≥ 1, p-adj < 0.05) were applied to ER (ERM) and mitochondrial (OMM) APEX-Seq data to facilitate comparison.…”

“…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.…”