Discovery of tsRNAs in mature sperm
In year 2012, by analyzing mature sperm small RNAs by RNA-seq in mice, we serendipitously found that the mature sperm contain a unique subset of tRNA–derived small RNAs (tsRNAs), mainly from 5′ transfer RNA halves and ranging in size from 29-34 nucleotides. tsRNAs show a drastic increase at late- spermatogenesis and post- spermatogenesis during epididymis maturation, suggesting regulated tRNA cleavage or/and selective concentrating mechanisms at these stages. tsRNAs are enriched in the sperm head and thus could be delivered into oocytes during fertilization. Like tRNAs, tsRNAs are highly conserved, leading us to propose that tsRNAs might serve as an ancient paternal element with evolutionarily conserved functions. (Cell Res 2012; Highlighted in Cell Res 2013)
Hidden information in sperm small RNAs: tRNA-derived small RNA (tsRNA) & rRNA-derived small RNA (rsRNA)
Discovery of tsRNAs in serum, across vertebrate species
After identifying tsRNAs as a new type of sperm small RNAs, we further discovered that tsRNAs are also abundantly and conservatively present in the serum of a wide range of vertebrates (from fish to human), and that serum tsRNAs are sensitive to pathological conditions (e.g. active infection) in mice, monkey and human beings. Importantly, we found that tsRNAs extracted from serum are more stable than chemically synthetic tsRNAs of the same sequence, suggesting RNA modifications in tsRNAs would contribute to their stabilization in the serum, thus represent another layer of information. (J Mol Cell Biol 2014)
Sperm tsRNAs/rsRNAs, and RNA modifications, contribute to epigenetic inheritance of paternally acquired traits
Parallel with the discovery of tsRNAs in sperm and serum, we are fascinated about the increasing evidence that certain acquired traits during paternal exposure can be “memorized” in the sperm and inherited by the offspring, leading to a resurrection of the ideas of 'Lamarckian inheritance' and 'Darwin's Pangenesis' (Nat Rev Genet 2016, Nat Rev Mol Cell Biol 2018). Recently, we found changes in expression profiles in sperm tsRNAs of mice with a paternal high-fat diet (HFD) (Science 2016), and later on also found another type of small RNAs, rRNA-derived small RNAs (rsRNAs), co-exist with tsRNAs in the sperm and they are sensitively regulated by HFD (Nat Cell Biol 2018). By injecting sperm total RNAs or different RNA fractions into normal zygotes, we found that the tsRNA/rsRNA-enriched 30-40nt sperm RNA fraction, along with associated RNA modifications, represent a carrier of paternal epigenetic information that contributes to intergenerational inheritance of diet-induced metabolic disorder (Science 2016; Nat Cell Biol 2018). We also demonstrated that a RNA methyltransferase, Dnmt2, can shape the sperm RNA ‘coding signature’ by regulating RNA modifications and tsRNA/rsRNA biogenesis, and affect RNAs’ secondary structures that may together contribute to the effect of epigenetic inheritance (Nat Cell Biol 2018).
Mammalian pre-implantation embryos provide a unique opportunity to study how a single cell diverges from a totipotent state into different fates within limited rounds of cell division. There are two longstanding questions about this process: When does the first blastomere-to-blastomere asymmetry emerge and how does this relate to the development of distinct cell fates? It remains debatable whether the first bifurcation of cell fate emerges randomly at the morula stage, or if it has been initiated at earlier stages before morphological differences arise. Combining single-cell RNA-seq analysis and mathematical modeling, we recently showed that the very first symmetry-breaking process involves both chance separation and defined transcriptional circuits (Development 2015; Highlighted in Development 2015), a new framework for future investigations.
Single-cell analysis: bridging randomness and determinism
From our single-embryo transcriptome analysis, small biases at molecular level will inevitably emerge at the 2-cell embryo stage, following a binomial distribution due to the cleavage division. At this stage, the blastomere-to-blastomere distribution seems random but during subsequent zygotic transcriptional activation, a “bistable pattern” emerges in some genes. Several lineage specifiers show a strong bias between different blastomeres thus providing potential for further increased asymmetry subsequently (Development 2015). These observations suggest a scenario of how order is created from a seemingly random process through the differential triggering of existing master regulators by the emergence of their small bias. As a triumph of our hypothesis, we further showed symmetry breaking driven by heterogeneous LincGET expression since 2-cell mouse embryo (Cell 2018).
Compartmentalized intracellular reactions: an alternative source of heterogeneity
The game of fate: deterministic & stochastic factors
in addition to the potential influence of stochastic events such as gene expression noise and uneven random segregation at cell division, we propose that cell-to-cell heterogeneity may also be initiated by pre-existing molecular inhomogeneity regulated by intracellular compartmentalization (Nat Commun 2018). The evidence for a spatially confined subcellular transcriptome together with the idea of compartmentalized reaction space provides a viable explanation of how mechanical cues, e.g. cell–cell contact during development, can alter transcriptional patterns and cell fate. In general, within a compartmentalized intracellular reaction space, the property of the space itself (volume and shape) has an essential influence on reaction rate. Morphological changes to a cell, either due to intrinsic factors such as physical constraints arising during development or due to external forces, could alter the reaction space. This would then lead to region-specific changes in biochemical reaction rates and products that could alter intracellular properties. It is possible that different cleavage division patterns of the mammalian embryo can result in differential cell–cell contact that affects blastomere geometry, which create differential reaction spaces and thus cell properties that bias cell fate (Nat Commun 2018)
PANDORA-seq (right optic) reads more hidden
'RNA code' than traditional RNA-seq (let optic)
Inspired by the movie: National Treasure
Symmetry-breaking in mammalian early embryo: when and how?
The concept of 'RNA Code' and how to decode it with advanced tools to promote precision medicine
Exploring the expanding world of small non-coding RNAs and their disease associations with new tools
We have also developed an LC-MS/MS based high-throughput platform that can simultaneously detect and quantify multiple types of RNA modifications in one RNA sample, initially used to study sperm RNA modifications (Science 2016, Nat Cell Biol 2018), and now being harnessed in other systems including for Alzheimer's disease (Neurobiol Dis 2020). By collaborating with Dr. Shenglong Zhang lab, we recently developed a mass spectrometry-based direct RNA sequencing method, MLC-Seq (J Am Chem Soc 2024) that can simultaneously unravel sequence and site-specific RNA modifications with stoichiometric precision, which can be used for examining tissue-specific differences in tRNAs/tsRNAs in regard to both sequence and modifications under normal and disease conditions.
To decode the RNA code and explore the expanding universe of small RNAs (Nat Cell Biol 2022), we developed a small RNA analyzing software SPORTS with Tong Zhou lab (GPB 2018) to facilitate the analyses of tsRNAs and rsRNAs and has led to a TRY-RNA signature for lung cancer diagnosis (Mol Cancer 2020); a new sequencing method PANDORA-seq to overcome RNA modifications that prevent small RNA detection in traditional RNA-seq (Nat Cell Biol 2021),
which uncovers a new and surprising small RNA landscape that is in fact, dominated by tsRNA/rsRNA, rather than miRNA in many tissues/cells. Interestingly, using PANDORA-seq, we found that tsRNAs are more enriched in the sperm heads compared to that of whole sperm, suggesting yet to be discovered function in the nuclei (Nat Cell Biol 2021).
Beyond RNAi: aptamer-like function of small RNAs
Our PANDORA-seq (Nat Cell Biol 2021) have unveiled a vastly expanded universe of small non-coding RNAs (sncRNAs) that extends far beyond the well-known miRNAs, discoveries showing that newly identified sncRNAs like tsRNAs and rsRNAs, along with their RNA modifications, regulate various cell functions and serve as carriers for epigenetic memory across generations. These tsRNAs and rsRNAs carry various RNA modifications, which can now be identified and mapped at nucleotide resolution with newly developed MLC-seq (J Am Chem Soc 2024). The RNA modifications are integral to the small RNA functionality and contribute to features beyond linear complementarity in a RNAi-like fashion, and to the 3D structures of these small RNAs, enable them to function in what we call an aptamer-like fashion (J Biol Chem 2023, Mol Cell 2023).
Fragmentation of tRNA give birth to tsRNAs with structural and fucntional potentials
Small RNA structure and modification in cell function
We posit that the yet-to-be-explored 3D structural features of these sncRNAs could be as crucial as their sequence-based roles in cellular regulation and epigenetic memory, a frontier we are actively exploring, aiming to uncover new layers of gene regulation.
With the increasing understandings of the information capacity mediated by sperm RNAs and RNA modifications and their involvement as novel layers of paternal hereditary information beyond DNA, we proposed the concept of sperm 'RNA code' (a combined RNA expression/modification signature) (Nat Rev Endocrinol 2019). We are exploring how the sperm RNA code interact with epigenetic reprogramming, and embryonic translational programming to control offspring phenotypes and how an RNA code can be harnessed for precision medicine, including the intergenerational prevention of metabolic diseases and cancer susceptibility.
