Single-cell profiling identifies LIN28A mRNA targets in the mouse pluripotent-to-2C-like transition and somatic cell...

Area of Science:

• Molecular Biology
• Stem Cell Biology
• Epigenetics

Background:

• RNA-binding proteins (RBPs) are critical regulators of cellular processes, including stem cell pluripotency and development.
• LIN28A is a key pluripotency regulator known to control microRNA maturation and mRNA translation/stability, but its mRNA targets in specific cell states remain incompletely understood.
• Understanding RBP-RNA interactions at a single-cell level is essential for deciphering complex biological processes like cell reprogramming and totipotency.

Purpose of the Study:

• To profile the mRNA targets of the RNA-binding protein LIN28A in mouse embryonic stem cells (ESCs), 2-cell embryo-like cells (2CLCs), and during somatic cell reprogramming.
• To elucidate the role of LIN28A in maintaining totipotency and facilitating pluripotency acquisition during cellular reprogramming.
• To investigate LIN28A-mediated RNA regulation at single-cell resolution.

Main Methods:

• Application of Targets of RBPs Identified By Editing (TRIBE) with single-cell resolution (scTRIBE) to map LIN28A-bound mRNAs.
• Quantitative single-cell analysis of scTRIBE datasets.
• Analysis of LIN28A-mRNA networks during somatic cell reprogramming.

Main Results:

• A significant increase in LIN28A binding to mRNAs encoding ribosome biogenesis factors and totipotency factors was observed in 2CLCs.
• LIN28A was found to extend the half-life of specific totipotency-related mRNAs, suggesting a role in maintaining the totipotent state.
• Distinct LIN28A-mRNA networks were identified in specific reprogramming trajectories, correlating with mesenchymal-to-epithelial transition and pluripotency acquisition.

Conclusions:

• LIN28A plays a multifaceted role in regulating stem cell states, extending beyond its known function in microRNA processing.
• LIN28A's interaction with specific mRNAs is crucial for maintaining totipotency and facilitating cellular reprogramming.
• Single-cell resolution of RNA-protein interactions provides critical insights into the dynamic regulation of gene expression in stem cells and reprogramming.