The transcriptome holds a wealth of information about the expression of genes and function of cells, which could hold key insights into embryonic development, aging, disease mechanisms and more. Current transcriptomics techniques are limited in their ability to simultaneously track RNA over both space and time, leaving many questions unanswered about the path transcripts take throughout their life cycle within a cell. Researchers from the Broad Institute of MIT and Harvard have developed a new method that could greatly expand the field of spatial transcriptomics, enabling the subcellular movement of RNA to be traced over time.
The researchers’ technique, called temporally resolved in situ sequencing and mapping (TEMPOmap), labels newly synthesized RNA and captures its location at specific timepoints through a combination of metabolic labeling, pulse-chase analysis and in situ RNA sequencing. This includes the use of a novel three-part chemical probe the specifically binds to metabolically labeled messenger RNA (mRNA), generates circular complementary DNA (cDNA) sequences and primes the cDNA for rolling cycle amplification (RCA), ensuring that only the labeled RNA is amplified and sequenced. TEMPOmap builds on STARmap (spatially-resolved transcript amplicon readout mapping) a technique previously developed by senior author Xiao Wang and colleagues that uses a hydrogel scaffold to anchor RNA molecules in 3D space at a specific point in time and an in situ sequencing approach called SEDAL (sequencing with error-reduction by dynamic annealing and ligation). Pulse-chase experiments with chase times ranging from 0 to 6 hours enabled the path of the labeled transcripts to be mapped over their lifetime.
TEMPOmap was used to map the movements of mRNAs transcribed from nearly 1,000 different genes in HeLa cells; the team also used the technique to track transcripts from dozens of genes in fibroblasts and cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) and more than 250 genes in human skin cells. The researchers were able to measure the speed of RNA transcription, nuclear export, movement throughout the cytoplasm and, ultimately, decay of the RNA molecules. The analyses showed that mRNAs transcribed from genes with specific functions in specific cell types tend to be synthesized faster and remain more stable in those cells than in other cell types. They also revealed differences in the kinetic patterns of mRNAs related to the molecular functions of the genes they encode. This research was published in Nature Methods.
“New ways of measuring the patterns of RNA movement and stability can provide fresh insights into basic principles of molecular biology, in addition to helping us better understand how RNA kinetics impact the function of cells across many different human cell types,” said Wang, who is a Merkin Institute Fellow at the Broad Institute and assistant professor of chemistry at MIT.
The research team aims to use their new spatiotemporal transcriptomics tool to monitor more biological processes, including by mapping the travel of gene transcripts in neurons to better understand brain function and dysfunction.