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Stanford Researchers Map RNA Localization Within Cells Using New Spatial Transcriptomics Method

NEW YORK (GenomeWeb) – A new spatial transcriptomics technique has allowed researchers from Stanford University to create an atlas of subcellular RNA localization with nanometer resolution, according to a new study.

Led by Howard Chang and Alice Ting, the researchers developed a method of RNA sequencing using direct proximity labeling with the peroxidase enzyme APEX2, dubbed APEX-seq. The researchers then used this method to reveal a map of RNA localization in nine distinct locales. They published their results Thursday in Cell.

"There are a lot of single-cell techniques in spatial transcriptomics that tell you localization across cells, but not within a cell," said Furqan Fazal, a postdoc at Stanford and first author of the paper. "We're trying something different, looking at localization within cells."

The Stanford Researchers said their method "should be applicable to any subcellular region and capture full sequence details of any RNA type, enabling comparisons across RNA variants and isoforms." Fazal added that the technique can be performed with reagents typically found in many labs studying transcriptomics.

They touted the fact that it provides full sequence information for RNA transcripts, that labeling is performed in living cells, and that it can "analyze 'unpurifiable' structures such as the nuclear lamina and outer membrane of the mitochondria that are impossible to access via fractionation-based approaches." The disadvantages of the method are that it cannot be used with human tissue, since it requires an APEX-fusion construct to be recombinantly expressed in the cell of interest, and it does not provide single-cell information.

APEX-seq is one of a growing number of technologies that combine spatial and molecular information. In the same issue of Cell, researchers from the Broad Institute described a method of single-molecule imaging and analysis that relies only on a next-generation sequencer for instrumentation. They join a number of other technologies, many of which look at RNA and the transcriptome, including seqFISH, Slide-seq, which was developed by different Broad researchers, and FISSEQ, developed in the lab of George Church of Harvard Medical School and the Wyss Institute.

The researchers said APEX-seq grew out of previous work using enzymes to map proteomes. The APEX2 enzyme, derived from soybeans, catalyzes biotin tagging "within a few nanometers of its active site in living cells" and had been previously combined with formaldehyde protein-RNA crosslinking to map organelles in the cell.

Here, the researchers simply figured out how to bypass crosslinking and directly tag cellular RNAs. They first validated the spatial specificity of this approach using RT-qPCR, then replaced that analysis with transcriptome-wide sequencing.

Having validated APEX-seq in the mitochondrial matrix and endoplasmic reticulum matrix, the researchers then looked at the seven other subcellular compartments. "The RNA content of most of these regions has not previously been mapped, as they are impossible to purify and/or are too small to image unambiguously by conventional microscopy," the authors wrote.

The lists of RNAs in the different locales suggested a few trends. Many of the RNAs had never been linked to the compartments suggested by APEX-seq (validated by sequential smFISH imaging.) "Most transcripts further localized to just one or two locations," and those locations can also be combined into four broad locations, the authors wrote. Messenger RNAs mostly localize to nuclear or cytosolic locations, while long non-coding RNAs mostly localize to nuclear locations, which is consistent with previous studies, they noted.

Fazal said the technique's chemical reaction is short, thus it would also allow him and other researchers to study "temporal dynamics" in the cell that take place over several minutes. "That's an important avenue of biology not studied greatly," he said.

The authors added in the paper that the newfound ability "to position endogenous RNAs with respect to distinct subcellular landmarks provides an exciting opportunity to test novel hypotheses concerning the relationship between RNA localization and function."