Tracing RNA biography: in situ transcriptome profiling by novel spatial omics technologies

Cell state and function are shaped by the spatiotemporal regulation of gene expression. This intricate pattern of gene expression is, in part, attained through the precise regulation of mRNA: its metabolism, transport, and translation within individual cells across spatial and temporal dimensions. Therefore, it is critical to methodically delineate the spatially resolved post-transcriptional regulations within transcriptomes, studying these events at the single-cell and single-molecule level. This advantage is important for mapping the complex network of transcriptional and post-transcriptional gene regulatory mechanisms inherent in cells and tissues. Moreover, our understanding of RNA translation in diverse cell types and states will be greatly enriched by the examination of spatially resolved protein synthesis patterns at the genomic scale within heterogeneous cells. Presently, the state-of-the-art spatial transcriptomic techniques offer only static snapshots of RNA expression, falling short of capturing RNA dynamics and their controlled translation within subcellular domains. Therefore, our driving question is whether the spatial regulation of multi-staged RNA life cycle influences cellular state and activity. Thus, an unmet need is to develop new methods capable of spatially tracking not only steady-state RNA expression but also their post-transcriptional states. This work is essential in providing a comprehensive picture of spatial RNA dynamics in cellular function and physiology. Filling this gap, I developed a novel in situ sequencing toolbox to study spatiallyresolved post-transcriptional RNA dynamics at the genomic scale in single cells during my PhD studies. My graduate work has led to the development of two novel in situ profiling technologies: (1) TEMPOmap (temporally resolved in situ sequencing and mapping), which resolves nascently-transcribed RNAs in space and time, and (2) RIBOmap (ribosome-bound mRNA mapping), a spatial ribosome profiling method. Utilizing these methods, we were able to holistically profile spatial, temporal and single-molecule information of RNA at the transcriptomic and translational levels in single cells. The main contribution of this work is that we established a specialized spatial transcriptomic toolkit specific for capturing the dynamics of mRNA in situ. Applying these technologies, I’ve profiled spatial, temporal and single-molecule information of RNA and single cells at the transcriptomic and translational levels in a range of biological systems, including iPSCs, primary skin cells and intact brain tissues. Specifically, I’ve focused on quantifying key steps in the mRNA life cycle in their spatial context, including RNA synthesis, nuclear export, translation, cytoplasmic translocation, and degradation. My goal was to better grasp the link between gene function and RNA lifespan at a genomic level across different cell types. Notably, we found that (1) different mRNAs are controlled both post-transcriptionally and translationally, with distinct subcellular localizations within cells; (2) in contrast to the previous belief that RNA dynamics solely depend on the primary sequence, they in fact exhibit diverse dynamic behavior for the same RNA species based on cell states, types, and even tissue regions. In primary skin samples, we noted cell-type-dependent alterations in the rates of RNA synthesis, transport, and degradation. Additionally, the translation level varied across cell types and regions within intact mouse brain tissue.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemistry

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2024

Author dc:creator

• Ren, Jingyi (Rena)

Advisor dc:contributor.advisor

• Wang, Xiao

Statement dc:rights

• Attribution-ShareAlike 4.0 International (CC BY-SA 4.0)
• Copyright retained by author(s)

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-sa/4.0/

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/157068

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/157068