Publications

Alternative polyadenylation (APA) has been discovered to play regulatory roles in the development of many cancer cells through preferential addition of a poly(A) tail at specific sites of pre-mRNA. A recent study found that APA was involved in the mediation of acute myeloid leukaemia (AML). However, unlike gene expression heterogeneity, little attention has been directed toward variations in single-cell APA for different cell types during AML development. Here, we used single-cell RNA-seq data of a massive population of 16,843 bone marrow mononuclear cells (BMMCs) from healthy and AML patient samples to investigate dynamic APA usage in different cell types. Abnormalities of APA dynamics in the BMMCs from AML patient samples were uncovered compared to the stable APA dynamics in samples from healthy individuals, as well as lower APA diversity between eight cell types in AML patients. Genes with APA dynamics specific to the AML samples were significantly enriched in cellular signal transduction pathways that contribute to AML development. Moreover, many leukaemic cell marker genes such as NF-κB, GATA2 and IAP-Family genes exhibited APA dynamics that specifically affected abnormal proliferation and differentiation of leukemic BMMCs. Additionally, mature erythroid cells displayed greater APA dynamics and global 3' UTR shortening compared with other cell types. Our results revealed extensive involvement of APA regulation in leukemia development and erythropoiesis at the single-cell level, providing a high-resolution atlas to navigate cellular mRNA processing landscapes of differentiated cells in AML.

Transcriptional networks are tightly controlled in plant development and stress responses. Alternative polyadenylation (APA) has been found to regulate gene expression under abiotic stress by increasing the heterogeneity at mRNA 3'-ends. Heavy metals like cadmium pollute water and soil due to mining and industry applications. Understanding how plants cope with heavy metal stress remains an interesting question. The Arabidopsis root hair was chosen as a single cell model to investigate the functional role of APA in cadmium stress response. Primary root growth inhibition and defective root hair morphotypes were observed. Poly(A) tag (PAT) libraries from single cell types, i.e., root hair cells, non-hair epidermal cells, and whole root tip under cadmium stress were prepared and sequenced. Interestingly, a root hair cell type-specific gene expression under short term cadmium exposure, but not related to the prolonged treatment, was detected. Differentially expressed poly(A) sites were identified, which largely contributed to altered gene expression, and enriched in pentose and glucuronate interconversion pathways as well as phenylpropanoid biosynthesis pathways. Numerous genes with poly(A) site switching were found, particularly for functions in cell wall modification, root epidermal differentiation, and root hair tip growth. Our findings suggest that APA plays a functional role as a potential stress modulator in root hair cells under cadmium treatment.

A crucial step for mRNA polyadenylation is poly(A) signal recognition by trans-acting factors. The mammalian cleavage and polyadenylation specificity factor (CPSF) complex components CPSF30 and WD repeat-containing protein33 (WDR33) recognize the canonical AAUAAA for polyadenylation. In Arabidopsis (Arabidopsis thaliana), the flowering time regulator FY is the homolog of WDR33. However, its role in mRNA polyadenylation is poorly understood. Using poly(A) tag sequencing, we found that >50% of alternative polyadenylation (APA) events are altered in fy single mutants or double mutants with oxt6 (a null mutant of AtCPSF30), but mutation of the FY WD40-repeat has a stronger effect than deletion of the plant-unique Pro-Pro-Leu-Pro-Pro (PPLPP) domain. fy mutations disrupt AAUAAA or AAUAAA-like poly(A) signal recognition. Notably, A-rich signal usage is suppressed in the WD40-repeat mutation but promoted in PPLPP-domain deficiency. However, fy mutations do not aggravate the altered signal usage in oxt6 Furthermore, the WD40-repeat mutation shows a preference for 3' untranslated region shortening, but the PPLPP-domain deficiency shows a preference for lengthening. Interestingly, the WD40-repeat mutant exhibits shortened primary roots and late flowering with alteration of APA of related genes. Importantly, the long transcripts of two APA genes affected in fy are related to abiotic stress responses. These results reveal a conserved and specific role of FY in mRNA polyadenylation.

Mangrove forests are an important contributor to the coastal marine environment. They have developed unique adaptations to the harsh coastal wetland, yet their geographic distribution is limited by environmental temperature. The adaptive strategies of mangrove at the molecular level, however, have not been addressed. In the present work, transcriptome analyses were performed on different cold damaged plants of a mangrove species, Kandelia obovata. From the samples collected in the field after a cold stress, we found that distinct expression profiles of many key genes are related to extreme temperature responses. These include transcription factors such as WRKY and bHLH, and other genes encoding proteins like SnRK2, PR-1, KCS, involving in the pathways of plant hormones, plant-pathogen interactions, and long chain fatty acid synthesis. We also examined the transcriptomes of eight tissues of K. obovata to identify candidate genes involved in adaptation and development. While stress-responsive genes were globally expressed, tissue-specific genes with diverse functions might be involved in tissue development and adaptability. For examples, genes encoding CYP724B1 and ABCB1 were specifically expressed in the fruit and root, respectively. Additionally, 26 genes were identified as positively selected genes in K. obovata, six of them were found to be involved in chilling stress response, seed germination and oxidation-reduction processes, suggesting their roles in stressful environment adaptation. Together, these results shed light into the K. obovata's natural responses to cold snaps at the molecular level, and reveal a global gene expression portrait across different tissues. It also provides a transcriptome resource for further molecular ecology studies and conservation planning of this and other mangrove plants in their native and adopted environments.

Genetic admixture, the intraspecific hybridization among divergent introduced sources, can immediately facilitate colonization via hybrid vigor and profoundly enhance invasion via contributing novel genetic variation to adaption. As hybrid vigor is short-lived, provisioning adaptation is anticipated to be the dominant and long-term profit of genetic admixture, but the evidence for this is rare. We employed the 30 years' geographic-scale invasion of the salt marsh grass, Spartina alterniflora, as an evolutionary experiment and evaluated the consequences of genetic admixture by combining the reciprocal transplant experiment with quantitative and population genetic surveys. Consistent with the documentation, we found that the invasive populations in China had multiple origins from the southern Atlantic coast and the Gulf of Mexico in the US. Interbreeding among these multiple sources generated a "hybrid swarm" that spread throughout the coast of China. In the northern and mid-latitude China, natural selection greatly enhanced fecundity, plant height and shoot regeneration compared to the native populations. Furthermore, genetic admixture appeared to have broken the negative correlation between plant height and shoot regeneration, which was genetically-based in the native range, and have facilitated the evolution of super competitive genotypes in the invasive range. In contrast to the evolved northern and mid-latitude populations, the southern invasive populations showed slight increase of plant height and shoot regeneration compared to the native populations, possibly reflecting the heterotic effect of the intraspecific hybridization. Therefore, our study suggests a critical role of genetic admixture in accelerating the geographic invasion via provisioning rapid adaptive evolution.

Alternative polyadenylation (APA) is an important way to regulate gene expression at the post-transcriptional level, and is extensively involved in plant stress responses. However, the systematic roles of APA regulation in response to abiotic and biotic stresses in rice at the genome scale remain unknown. To take advantage of available RNA-seq datasets, using a novel tool APAtrap, we identified thousands of genes with significantly differential usage of polyadenylation [poly(A)] sites in response to the abiotic stress (drought, heat shock, and cadmium) and biotic stress [bacterial blight (BB), rice blast, and rice stripe virus (RSV)]. Genes with stress-responsive APA dynamics commonly exhibited higher expression levels when their isoforms with short 3' untranslated region (3' UTR) were more abundant. The stress-responsive APA events were widely involved in crucial stress-responsive genes and pathways: e.g. APA acted as a negative regulator in heat stress tolerance; APA events were involved in DNA repair and cell wall formation under Cd stress; APA regulated chlorophyll metabolism, being associated with the pathogenesis of leaf diseases under RSV and BB challenges. Furthermore, APA events were found to be involved in glutathione metabolism and MAPK signaling pathways, mediating a crosstalk among the abiotic and biotic stress-responsive regulatory networks in rice. Analysis of large-scale datasets revealed that APA may regulate abiotic and biotic stress-responsive processes in rice. Such post-transcriptome diversities contribute to rice adaption to various environmental challenges. Our study would supply useful resource for further molecular assisted breeding of multiple stress-tolerant cultivars for rice.

The adaptive value of transgenerational effects (the ancestor environmental effects on offspring) in changing environments has received much attention in recent years, but the related empirical evidence remains equivocal. Here, we conducted a meta-analysis summarising 139 experimental studies in plants and animals with 1170 effect sizes to investigate the generality of transgenerational effects across taxa, traits, and environmental contexts. It was found that transgenerational effects generally enhanced offspring performance in response to both stressful and benign conditions. The strongest effects are in annual plants and invertebrates, whereas vertebrates appear to benefit mostly under benign conditions, and perennial plants show hardly any transgenerational responses at all. These differences among taxonomic/life-history groups possibly reflect that vertebrates can avoid stressful conditions through their mobility, and longer-lived plants have alternative strategies. In addition to environmental contexts and taxonomic/life-history groups, transgenerational effects also varied among traits and developmental stages of ancestors and offspring, but the effects were similarly strong across three generations of offspring. By way of a more comprehensive data set and a different effect size, our results differ from those of a recent meta-analysis, suggesting that transgenerational effects are widespread, strong and persistent and can substantially impact the responses of plants and animals to changing environments.

Auxin is widely involved in plant growth and development. However, the molecular mechanism on how auxin carries out this work is unclear. In particular, the effect of auxin on pre-mRNA post-transcriptional regulation is mostly unknown. By using a poly(A) tag (PAT) sequencing approach, mRNA alternative polyadenylation (APA) profiles after auxin treatment were revealed. We showed that hundreds of poly(A) site clusters (PACs) are affected by auxin at the transcriptome level, where auxin reduces PAC distribution in 5'-untranslated region (UTR), but increases in the 3'UTR. APA site usage frequencies of 42 genes were switched by auxin, suggesting that auxin affects the choice of poly(A) sites. Furthermore, poly(A) signal selection was altered after auxin treatment. For example, a mutant of poly(A) signal binding protein CPSF30 showed altered sensitivity to auxin treatment, indicating interactions between auxin and the poly(A) signal recognition machinery. We also found that auxin activity on lateral root development is likely mediated by altered expression of ARF7, ARF19 and IAA14 through poly(A) site switches. Our results shed light on the molecular mechanisms of auxin responses relative to its interactions with mRNA polyadenylation.

Mangrove plants adapt to coastal tidal mudflats with specially evolved viviparity seed development. However, very little is known about the genetic and molecular mechanisms of mangrove viviparity. Here, we tested a hypothesis that plant hormone abscisic acid (ABA) plays a significant role in precocious germination of viviparous Kandelia obovata seeds by exogenous applications. Through transcriptome analysis of ABA treated seeds, it was found that ABA repressed mangrove fruit growth and development, and there were thousands of genes differentially expressed. As a result, dynamics of the pathways were dramatically altered. In particular, "Plant hormone signal transduction" and "MAPK signaling pathway" were represented significantly. Among differentially expressed genes, some key genes of ABA signal transduction were induced, while ABA biosynthesis genes were repressed. Take ABI1 and ABI2, key negative regulators in ABA signal pathway, as examples, homologous alignment and a phylogenetic tree in various species showed that ABI1 and ABI2 are highly conserved among various species. The functional similarity of these genes was confirmed by transgenic work in Arabidopsis. Taken together, ABA inhibited mangrove viviparity, but mangroves developed a mechanism to prevent accidently increase of ABA in the harsh environment for maintaining viviparous reproductive strategy.

MOTIVATION: Alternative polyadenylation (APA) has been increasingly recognized as a crucial mechanism that contributes to transcriptome diversity and gene expression regulation. As RNA-seq has become a routine protocol for transcriptome analysis, it is of great interest to leverage such unprecedented collection of RNA-seq data by new computational methods to extract and quantify APA dynamics in these transcriptomes. However, research progress in this area has been relatively limited. Conventional methods rely on either transcript assembly to determine transcript 3' ends or annotated poly(A) sites. Moreover, they can neither identify more than two poly(A) sites in a gene nor detect dynamic APA site usage considering more than two poly(A) sites.

RESULTS: We developed an approach called APAtrap based on the mean squared error model to identify and quantify APA sites from RNA-seq data. APAtrap is capable of identifying novel 3' UTRs and 3' UTR extensions, which contributes to locating potential poly(A) sites in previously overlooked regions and improving genome annotations. APAtrap also aims to tally all potential poly(A) sites and detect genes with differential APA site usages between conditions. Extensive comparisons of APAtrap with two other latest methods, ChangePoint and DaPars, using various RNA-seq datasets from simulation studies, human and Arabidopsis demonstrate the efficacy and flexibility of APAtrap for any organisms with an annotated genome.

AVAILABILITY AND IMPLEMENTATION: Freely available for download at https://apatrap.sourceforge.io.

CONTACT: liqq@xmu.edu.cn or xhuister@xmu.edu.cn.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.