Abstract
The spatial arrangement of newly synthesized transcriptome in eukaryotic cells underlies various biological processes including cell proliferation and differentiation. In this study, we combine metabolic incorporation of electron-rich ribonucleosides (e.g., 6-thioguanosine and 4-thiouridine) with a peroxidase-mediated proximity-dependent RNA labeling technique (APEX-seq) to develop a sensitive method, termed MERR APEX-seq, for selectively profiling newly transcribed RNAs at specific subcellular locations in live cells. We demonstrate that MERR APEX-seq is 20-fold more efficient than APEX-seq and offers both high spatial specificity and high coverage in mitochondrial matrix. At the ER membrane, 91% of the transcripts captured by MERR APEX-seq encode for secretory pathway proteins, thus demonstrating the high spatial specificity of MERR APEX-seq in open subcellular compartments. Application of MERR APEX-seq to the nuclear lamina of human cells reveals a local transcriptome of 1,012 RNAs, many of which encode for nuclear proteins involved in histone modification, chromosomal structure maintenance, and RNA processing.
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