Publications

MOTIVATION: Alternative polyadenylation (APA) plays a key post-transcriptional regulatory role in mRNA stability and functions in eukaryotes. Single cell RNA-seq (scRNA-seq) is a powerful tool to discover cellular heterogeneity at gene expression level. Given 3' enriched strategy in library construction, the most commonly used scRNA-seq protocol-10× Genomics enables us to improve the study resolution of APA to the single cell level. However, currently there is no computational tool available for investigating APA profiles from scRNA-seq data.

RESULTS: Here, we present a package scDAPA for detecting and visualizing dynamic APA from scRNA-seq data. Taking bam/sam files and cell cluster labels as inputs, scDAPA detects APA dynamics using a histogram-based method and the Wilcoxon rank-sum test, and visualizes candidate genes with dynamic APA. Benchmarking results demonstrated that scDAPA can effectively identify genes with dynamic APA among different cell groups from scRNA-seq data.

AVAILABILITY AND IMPLEMENTATION: The scDAPA package is implemented in Shell and R, and is freely available at https://scdapa.sourceforge.io.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Calpains represent a family of calcium-dependent proteases participating in a multitude of functions under physiological or pathological conditions. Calpain-1 is one of the most studied members of the family, is ubiquitously distributed in organs and tissues, and has been shown to be involved in synaptic plasticity and neuroprotection in mammalian brain. Calpain-1 deletion results in a number of phenotypic alterations. While some of these alterations can be explained by the acute functions of calpain-1, the present study was directed at studying alterations in gene expression that could also account for these phenotypic modifications. RNA-seq analysis identified 354 differentially expressed genes (DEGs) in brain of calpain-1 knock-out mice, as compared to their wild-type strain. Most DEGs were classified in 10 KEGG pathways, with the highest representations in Protein Processing in Endoplasmic Reticulum, MAP kinase and Alzheimer's disease pathways. Most DEGs were down-regulated and validation of a number of these genes indicated a corresponding decreased expression of their encoded proteins. The results indicate that calpain-1 is involved in the regulation of a significant number of genes affecting multiple brain functions. They also indicate that mutations in calpain-1 are likely to be involved in a number of brain disorders.

Poly(A) tail length (PAL) has been implicated in the regulation of mRNA translation activities. However, the extent of such regulation at the transcriptome level is less understood in plants. Herein, we report the development and optimization of a large-scale sequencing technique called the Assay for PAL-sequencing (APAL-seq). To explore the role of PAL on post-transcriptional modification and translation, we performed PAL profiling of Arabidopsis transcriptome in response to heat shock. Transcripts of 2,477 genes were found to have variable PAL upon heat treatments. Further study of the transcripts of 14 potential heat-responsive genes identified two distinct groups of genes. In one group, PAL was heat stress-independent, and in the other, PAL was heat stress-sensitive. Meanwhile, the protein expression of HSP70 and HSP17.6C was determined to test the impact of PAL on translational activity. In the absence of heat stress, neither gene demonstrated protein expression; however, under gradual or abrupt heat stress, both transcripts showed enhanced protein expression with elongated PAL. Interestingly, HSP17.6C protein levels were positively correlated with the severity of heat treatment and peaked when treated with abrupt heat. Our results suggest that plant genes have a high variability of PALs and that PAL contributes to swift posttranslational stress responses.

Alternative polyadenylation (APA) occurs in the process of mRNA maturation by adding a poly(A) tail at different locations, resulting increased diversity of mRNA isoforms and contributing to the complexity of gene regulatory network. Benefit from the development of high-throughput sequencing technologies, we could now delineate APA profiles of transcriptomes at an unprecedented pace. Especially the single cell RNA sequencing (scRNA-seq) technologies provide us opportunities to interrogate biological details of diverse and rare cell types. Despite increasing evidence showing that APA is involved in the cell type-specific regulation and function, efficient and specific laboratory methods for capturing poly(A) sites at single cell resolution are underdeveloped to date. In this review, we summarize existing experimental and computational methods for the identification of APA dynamics from diverse single cell types. A future perspective is also provided.

Eukaryotic histone deacetylation, critical for maintaining nucleosome structure and regulating gene expression, is mediated by histone deacetylases (HDACs). Although nucleosomes have been reported to regulate mRNA polyadenylation in humans, the role of HDACs in regulating polyadenylation has not been uncovered. Taking advantage of phenotypic studies on Arabidopsis, HDA6 (one of HDACs) was found to be a critical part of many biological processes. Here, we report that HDA6 affects mRNA polyadenylation in Arabidopsis Poly(A) sites of up-regulated transcripts are closer to the histone acetylation peaks in hda6 compared to the wild-type Col-0. HDA6 is required for the deacetylation of histones around DNA on nucleosomes, which solely coincides with up-regulated or uniquely presented poly(A) sites in hda6 Furthermore, defective HDA6 results in an overrepresentation of the canonical poly(A) signal (AAUAAA) usage. Chromatin loci for generating AAUAAA-type transcripts have a comparatively low H3K9K14ac around poly(A) sites when compared to other noncanonical poly(A) signal-containing transcripts. These results indicate that HDA6 regulates polyadenylation in a histone deacetylation-dependent manner in Arabidopsis.

The comment by Sánchez-Tójar et al. (2020, Ecol Lett) questioned the methodology, transparency and conclusion of our study (Ecol Lett, 22, 2019, 1976). The comment has overlooked important evolutionary assumptions in their reanalysis, and the issues raised were in fact dealt with through the peer-review process. Far from being biased, the key conclusion of our meta-analysis still stands; transgenerational effects are largely adaptive.

Large amounts of wastewater are generated from stone processing, which are toxic and cause serious environmental and health risks. To quantify the content of stone processing wastewater and estimate its effects on plant growth, we collected water samples from sewage outfall of four stone processing factories and nearby water bodies. The concentration of potential toxic metals were much higher in the wastewater than background controls. Wastewater inhibited plant primary root elongation, lateral root formation, and growth of aerial part. Seedlings treated with the effluents were unhealthy with deep purple leaves and usually died before flowering. Chlorophyll a/b contents and chloroplast number were reduced in those abnormal mesophyll cells. Transcriptional levels were decreased for chloroplast formation genes, but increased for those participated in chloroplast degradation and catabolism. Six out of nine tested senescence-associated genes were up-regulated. Furthermore, our results show that endogenous toxic metal levels indeed increased after wastewater treatment. Altogether, these results indicated that the potential toxic metals rich wastewater had significant inhibition on plant growth and led to senescence-associated program cell death, which could be helpful for the government and enterprises to understand the environmental risks and formulate reasonable wastewater emission standards for the stone processing industry.

Alternative polyadenylation (APA) is a widespread post-transcriptional mechanism that regulates gene expression through mRNA metabolism, playing a pivotal role in modulating phenotypic traits in rice (Oryza sativa L.). However, little is known about the APA-mediated regulation underlying the distinct characteristics between two major rice subspecies, indica and japonica. Using a poly(A)-tag sequencing approach, polyadenylation (poly(A)) site profiles were investigated and compared pairwise from germination to the mature stage between indica and japonica, and extensive differentiation in APA profiles was detected genome-wide. Genes with subspecies-specific poly(A) sites were found to contribute to subspecies characteristics, particularly in disease resistance of indica and cold-stress tolerance of japonica. In most tissues, differential usage of APA sites exhibited an apparent impact on the gene expression profiles between subspecies, and genes with those APA sites were significantly enriched in quantitative trait loci (QTL) related to yield traits, such as spikelet number and 1000-seed weight. In leaves of the booting stage, APA site-switching genes displayed global shortening of 3' untranslated regions with increased expression in indica compared with japonica, and they were overrepresented in the porphyrin and chlorophyll metabolism pathways. This phenomenon may lead to a higher chlorophyll content and photosynthesis in indica than in japonica, being associated with their differential growth rates and yield potentials. We further constructed an online resource for querying and visualizing the poly(A) atlas in these two rice subspecies. Our results suggest that APA may be largely involved in developmental differentiations between two rice subspecies, especially in leaf characteristics and the stress response, broadening our knowledge of the post-transcriptional genetic basis underlying the divergence of rice traits.

Pre-mRNA cleavage and polyadenylation is an essential step for almost all mRNA in eukaryotes. The cis-elements around the poly(A) sites, however, are very diverse among different organisms. We characterized the poly(A) signals of seven different species, and compared them with that of four well-studied organisms. We found that ciliates do not show any dominant poly(A) signal; a triplet (UAA) and tetramers (UAAA and GUAA) are dominant in diatoms and red alga, respectively; and green alga Ostreococcus uses UGUAA as its poly(A) signal. Spikemoss and moss use conserved AAUAAA signals that are similar to other land plants. Our analysis suggests that the first two bases (NN in NNUAAA) are likely degenerated whereas UAAA appears to be the core motif. Combined with other published results, it is suggested that the highly conserved poly(A) signal AAUAAA may be derived from UAA with an intermediate, putative UAAA, following a pathway of UAA→UAAA→AAUAAA.

The post-transcriptional regulation involved in the responses of diatoms to silicon is poorly understood. Using a poly(A)-tag sequencing (PAT-seq) technique that interrogates only the junctions of 3'-untranslated region (UTR) and the poly(A) tails at the transcriptome level, a comprehensive comparison of alternative polyadenylation (APA) was performed to understand the role of post-transcriptional regulation in various silicon-related cellular responses for the marine diatom Thalassiosira pseudonana. In total, 23 701 poly(A) clusters and 6894 APA genes, treated with silicon starvation and replenishment, were identified at nine time points. Significant APA was found in numerous genes (e.g. five cingulin genes) closely associated with the silicon-starvation response, girdle bands and valve synthesis, suggesting that many genes participated in the responses to silicon availability and biosilica formation through changes in transcript isoforms. The poly(A) site usage profiles were distinct during various stages of silicon biomineralization responses. Moreover, a correlation between APA and expression levels of APA switching genes was also discovered. This is an interesting study that presents a genome-wide profile of transcript ends in diatoms, which is distinct from that of higher plants, animals and other microalgae. This work provides an important resource to understand a different aspect of cell-wall synthesis.