Research

microscope

1. Mechanisms of mRNA polyadenylation

mRNA polyadenylation is a crucial step in eukaryotic gene expression, influencing mRNA stability, translatability, and cytoplasmic translocation. This process also impacts pre-mRNA splicing and transcription termination. Recent research has linked mRNA polyadenylation to various cellular processes, including responses to environmental stimuli, pathogen invasion, and cancer development.

Our lab aims to uncover the molecular mechanisms of mRNA polyadenylation at genetic, transcriptomic, and genomic levels, examining both tissue-specific and single-cell contexts. We use functional genomic tools to study these mechanisms in model plants such as Arabidopsis and rice, focusing on the biochemical, genetic, and molecular functions of polyadenylation factors and their roles in plant gene expression during development and stress responses.

Leveraging advanced RNA-sequencing technologies, we conduct large-scale analyses of alternative polyadenylation (APA) to map the comprehensive landscape of APA across single cells and entire organisms. With over 70% of genes utilizing APA for expression regulation, the role of APA in cellular and organismal functions is just beginning to be understood. Our multi-omics approach reveals that APA is linked to various cellular processes, including circadian regulation, epigenetic control, and quantitative traits, highlighting its role as a central hub for fine-tuning gene expression at the transcriptome level.

Test tubes

2. Gene expression in neuronal cell communication

Our research collaboration extends to gene expression regulation in human induced pluripotent stem cells (iPSCs), particularly in neuronal disease models like Alzheimer’s disease (AD). AD and related dementias present a global challenge, characterized by extensive neuronal loss, yet the mechanisms behind this loss remain unclear.

Microglia, the central nervous system’s resident immune cells, are suspected to play a role in AD pathogenesis. We explore how extracellular vesicles (EVs) — lipid bilayer membrane vesicles 30 to 200 nm in diameter, generated by the endosomal system — influence cell-to-cell communication. EVs contain proteins, mRNA, and miRNA that can modulate gene expression in recipient cells. Notably, microglia-derived microvesicles have been shown to regulate neuronal excitability, representing a promising area of research in neurodegenerative diseases.

We employ multi-omics tools to analyze the components of microglia-derived EVs in AD-like models derived from patient and control samples. Identifying these potential markers and their roles in regulating gene expression, especially concerning neuronal degeneration, could provide new insights into neuronal gene expression regulation and identify potential targets for future drug development.

Lab bench

3. Gene expression regulation in development and responses

In collaboration with various research groups, we study gene function and expression related to plant development and responses to environmental stimuli.

Mangroves, for example, are woody plants that thrive in extreme coastal conditions. Their unique adaptations, such as viviparous seed reproduction, pneumatophores for submergence and hypoxia, and various salinity responses, are of significant interest due to their potential applications in crop breeding programs. We investigate these adaptations through gene expression analysis to understand the molecular mechanisms underlying these features.

Additionally, we explore transgenerational transmission of parental phenotypes and epigenetic modifications, which may offer new insights into how organisms adapt to acute stresses and how such “memories” are passed to future generations. Our past research also includes efforts in plant disease resistance through genetic engineering of antimicrobial peptides to improve crop resilience.

Publications

  • Lin X, Yin J, Wang Y, Yao J, Li QQ, Latzel V, Bossdorf O, Zhang Y-Y. Environment-induced heritable variations are common in Arabidopsis thaliana.. Nature communications. 2024;15(1):4615. doi:10.1038/s41467-024-49024-3

    Parental or ancestral environments can induce heritable phenotypic changes, but whether such environment-induced heritable changes are a common phenomenon remains unexplored. Here, we subject 14 genotypes of Arabidopsis thaliana to 10 different environmental treatments and observe phenotypic and genome-wide gene expression changes over four successive generations. We find that all treatments caused heritable phenotypic and gene expression changes, with a substantial proportion stably transmitted over all observed generations. Intriguingly, the susceptibility of a genotype to environmental inductions could be predicted based on the transposon abundance in the genome. Our study thus challenges the classic view that the environment only participates in the selection of heritable variation and suggests that the environment can play a significant role in generating of heritable variations.

  • Lin J, Li QQ. Coupling epigenetics and RNA polyadenylation: missing links.. Trends in plant science. 2023;28(2):223–234. doi:10.1016/j.tplants.2022.08.023

    Precise regulation of gene expression is crucial for plant survival. As a cotranscriptional regulatory mechanism, pre-mRNA polyadenylation is essential for fine-tuning gene expression. Polyadenylation can be alternatively projected at various sites of a transcript, which contributes to transcriptome diversity. Epigenetic modification is another mechanism of transcriptional control. Recent studies have uncovered crosstalk between cotranscriptional polyadenylation processes and both epigenomic and epitranscriptomic markers. Genetic analyses have demonstrated that DNA methylation, histone modifications, and epitranscriptomic modification are involved in regulating polyadenylation in plants. Here we summarize current understanding of the links between epigenetics and polyadenylation and their novel biological efficacy for plant development and environmental responses. Unresolved issues and future directions are discussed to shed light on the field.

  • Hong Y, Dong X, Chang L, Xie C, Chang M, Aguilar JS, Lin J, Lin J, Li QQ. Microglia-containing cerebral organoids derived from induced pluripotent stem cells for the study of neurological diseases.. iScience. 2023;26(3):106267. doi:10.1016/j.isci.2023.106267

    Microglia play an important role in neuroinflammation and neurodegeneration. Here, we report an approach for generating microglia-containing cerebral organoids derived from human pluripotent stem cells involving the supplementation of growth factors (FGF, EGF, heparin) and 10% CO2 culture conditions. Using this platform, Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS-PDC) cerebral organoids were generated from patient-derived induced pluripotent stem cells (iPSCs). These ALS-PDC-affected organoids had more reactive astrocytes and M1 microglia, and had fewer M2 microglia than their unaffected counterparts, leading to impaired microglia-mediated phagocytosis. RNA-seq analysis of ALS-PDC and control organoids indicated that the most significant changes were microglia- and astrocyte-related genes (IFITM1/2, TGF-β, and GFAP). The most significantly downregulated pathway was type I interferon signaling. Interferon-gamma supplementation increased IFITM expression, enhanced microglia-mediated phagocytosis, and reduced beta-amyloid accumulation in ALS-PDC-affected network. The results demonstrated the feasibility of using microglia-containing organoids for the study of neurodegenerative diseases.

  • He N, Huang F, Lu L, Wang X, Li QQ, Yang D. SPR9 encodes a 60 S ribosomal protein that modulates panicle spreading and affects resistance to false smut in rice (Oryza sativa. L).. BMC plant biology. 2023;23(1):205. doi:10.1186/s12870-023-04172-4

    BACKGROUND: The architecture of inflorescence in crops is a key agronomic feature determining grain yield and thus has been a major target trait of cereal domestication.

    RESULTS: In this study, we show that a simple spreading panicle change in rice panicle shape, controlled by the Spreading Panicle 9 (SPR9) locus, also has a significant impact on the resistance to rice false smut (RFS). Meanwhile, we mapped a novel spr9 mutant gene between markers Indel5-18 and Indel5-22 encompassing a genomic region of 43-kb with six candidate genes. Through gene prediction and cDNA sequencing, we confirmed that LOC_Os05g38520 is the target gene in the spr9 mutant, which encodes 60 S ribosomal protein L36-2. Further analysis showed that the spr9 mutant is caused by a 1 bp deletion in the first exon that resulted in premature termination. Knockout experiments showed that the SPR9 gene is responsible for the spreading panicle phenotype of the spr9 mutant. Interestingly, the spr9 mutant was found to improve resistance to RFS without affecting major agronomic traits. Taken together, our results revealed that the spr9 allele has good application prospects in rice breeding for disease resistance and panicle improvement.

    CONCLUSIONS: We report the map-based cloning and functional characterization of SPR9, which encodes a 60 S ribosomal protein that regulates spreading panicles and affects the resistance to false smut in rice.

  • Ma H, Lin J, Mei F, Mao H, Li QQ. Differential alternative polyadenylation of homoeologous genes of allohexaploid wheat ABD subgenomes during drought stress response.. The Plant journal : for cell and molecular biology. 2023;114(3):499–518. doi:10.1111/tpj.16150

    Because allohexaploid wheat genome contains ABD subgenomes, how the expression of homoeologous genes is coordinated remains largely unknown, particularly at the co-transcriptional level. Alternative polyadenylation (APA) is an important part of co-transcriptional regulation, which is crucial in developmental processes and stress responses. Drought stress is a major threat to the stable yield of wheat. Focusing on APA, we used poly(A) tag sequencing to track poly(A) site dynamics in wheat under drought stress. The results showed that drought stress led to extensive APA involving 37-47% of differentially expressed genes in wheat. Significant poly(A) site switching was found in stress-responsive genes. Interestingly, homoeologous genes exhibit unequal numbers of poly(A) sites, divergent APA patterns with tissue specificity and time-course dynamics, and distinct 3'-UTR length changes. Moreover, differentially expressed transcripts in leaves and roots used different poly(A) signals, the up- and downregulated isoforms had distinct preferences for non-canonical poly(A) sites. Genes that encode key polyadenylation factors showed differential expression patterns under drought stress. In summary, poly(A) signals and the changes in core poly(A) factors may widely affect the selection of poly(A) sites and gene expression levels during the response to drought stress, and divergent APA patterns among homoeologous genes add extensive plasticity to this responsive network. These results not only reveal the significant role of APA in drought stress response, but also provide a fresh perspective on how homoeologous genes contribute to adaptability through transcriptome diversity. In addition, this work provides information about the ends of transcripts for a better annotation of the wheat genome.

  • Hao S, Zhang L, Zhao D, Zhou J, Ye C, Qu H, Li QQ. Inhibitor AN3661 reveals biological functions of Arabidopsis CLEAVAGE and POLYADENYLATION SPECIFICITY FACTOR 73.. Plant physiology. 2023;193(1):537–554. doi:10.1093/plphys/kiad352

    Cleavage and polyadenylation specificity factor (CPSF) is a protein complex that plays an essential biochemical role in mRNA 3'-end formation, including poly(A) signal recognition and cleavage at the poly(A) site. However, its biological functions at the organismal level are mostly unknown in multicellular eukaryotes. The study of plant CPSF73 has been hampered by the lethality of Arabidopsis (Arabidopsis thaliana) homozygous mutants of AtCPSF73-I and AtCPSF73-II. Here, we used poly(A) tag sequencing to investigate the roles of AtCPSF73-I and AtCPSF73-II in Arabidopsis treated with AN3661, an antimalarial drug with specificity for parasite CPSF73 that is homologous to plant CPSF73. Direct seed germination on an AN3661-containing medium was lethal; however, 7-d-old seedlings treated with AN3661 survived. AN3661 targeted AtCPSF73-I and AtCPSF73-II, inhibiting growth through coordinating gene expression and poly(A) site choice. Functional enrichment analysis revealed that the accumulation of ethylene and auxin jointly inhibited primary root growth. AN3661 affected poly(A) signal recognition, resulted in lower U-rich signal usage, caused transcriptional readthrough, and increased the distal poly(A) site usage. Many microRNA targets were found in the 3' untranslated region lengthened transcripts; these miRNAs may indirectly regulate the expression of these targets. Overall, this work demonstrates that AtCPSF73 plays important part in co-transcriptional regulation, affecting growth, and development in Arabidopsis.

  • Zhou J, Li QQ. Stress responses of plants through transcriptome plasticity by mRNA alternative polyadenylation.. Molecular horticulture. 2023;3(1):19. doi:10.1186/s43897-023-00066-z

    The sessile nature of plants confines their responsiveness to changing environmental conditions. Gene expression regulation becomes a paramount mechanism for plants to adjust their physiological and morphological behaviors. Alternative polyadenylation (APA) is known for its capacity to augment transcriptome diversity and plasticity, thereby furnishing an additional set of tools for modulating gene expression. APA has also been demonstrated to exhibit intimate associations with plant stress responses. In this study, we review APA dynamic features and consequences in plants subjected to both biotic and abiotic stresses. These stresses include adverse environmental stresses, and pathogenic attacks, such as cadmium toxicity, high salt, hypoxia, oxidative stress, cold, heat shock, along with bacterial, fungal, and viral infections. We analyzed the overarching research framework employed to elucidate plant APA response and the alignment of polyadenylation site transitions with the modulation of gene expression levels within the ambit of each stress condition. We also proposed a general APA model where transacting factors, including poly(A) factors, epigenetic regulators, RNA m6A modification factors, and phase separation proteins, assume pivotal roles in APA related transcriptome plasticity during stress response in plants.

  • Salt tolerance is an important mechanism by which plants can adapt to a saline environment. To understand the process of salt tolerance, we performed global analyses of mRNA alternative polyadenylation (APA), an important regulatory mechanism during eukaryotic gene expression, in Arabidopsis thaliana and its halophytic relative Eutrema salsugineum with regard to their responses to salt stress. Analyses showed that while APA occurs commonly in both Arabidopsis and Eutrema, Eutrema possesses fewer APA genes than Arabidopsis (47% vs. 54%). However, the proportion of APA genes was significantly increased in Arabidopsis under salt stress but not in Eutrema. This indicated that Arabidopsis is more sensitive to salt stress and that Eutrema exhibits an innate response to such conditions. Both species utilized distal poly(A) sites under salt stress; however, only eight genes were found to overlap when their 3' untranslated region (UTR) lengthen genes were compared, thus revealing their distinct responses to salt stress. In Arabidopsis, genes that use distal poly(A) sites were enriched in response to salt stress. However, in Eutrema, the use of poly(A) sites was less affected and fewer genes were enriched. The transcripts with upregulated poly(A) sites in Arabidopsis showed enriched pathways in plant hormone signal transduction, starch and sucrose metabolism, and fatty acid elongation; in Eutrema, biosynthetic pathways (stilbenoid, diarylheptanoid, and gingerol) and metabolic pathways (arginine and proline) showed enrichment. APA was associated with 42% and 29% of the differentially expressed genes (DE genes) in Arabidopsis and Eutrema experiencing salt stress, respectively. Salt specific poly(A) sites and salt-inducible APA events were identified in both species; notably, some salt tolerance-related genes and transcription factor genes exhibited differential APA patterns, such as CIPK21 and LEA4-5. Our results suggest that adapted species exhibit more orderly response at the RNA maturation step under salt stress, while more salt-specific poly(A) sites were activated in Arabidopsis to cope with salinity conditions. Collectively, our findings not only highlight the importance of APA in the regulation of gene expression in response to salt stress, but also provide a new perspective on how salt-sensitive and salt-tolerant species perform differently under stress conditions through transcriptome diversity.