Differential alternative polyadenylation of homoeologous genes of allohexaploid wheat ABD subgenomes during drought stress response.

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.

Inhibitor AN3661 reveals biological functions of Arabidopsis CLEAVAGE and POLYADENYLATION SPECIFICITY FACTOR 73.

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.

HDA6-dependent histone deacetylation regulates mRNA polyadenylation in Arabidopsis.

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.

SPR9 encodes a 60 S ribosomal protein that modulates panicle spreading and affects resistance to false smut in rice (Oryza sativa. L).

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.

QuantifyPoly(A): reshaping alternative polyadenylation landscapes of eukaryotes with weighted density peak clustering.

The dynamic choice of different polyadenylation sites in a gene is referred to as alternative polyadenylation, which functions in many important biological processes. Large-scale messenger RNA 3' end sequencing has revealed that cleavage sites for polyadenylation are presented with microheterogeneity. To date, the conventional determination of polyadenylation site clusters is subjective and arbitrary, leading to inaccurate annotations. Here, we present a weighted density peak clustering method, QuantifyPoly(A), to accurately quantify genome-wide polyadenylation choices. Applying QuantifyPoly(A) on published 3' end sequencing datasets from both animals and plants, their polyadenylation profiles are reshaped into myriads of novel polyadenylation site clusters. Most of these novel polyadenylation site clusters show significantly dynamic usage across different biological samples or associate with binding sites of trans-acting factors. Upstream sequences of these clusters are enriched with polyadenylation signals UGUA, UAAA and/or AAUAAA in a species-dependent manner. Polyadenylation site clusters also exhibit species specificity, while plants ones generally show higher microheterogeneity than that of animals. QuantifyPoly(A) is broadly applicable to any types of 3' end sequencing data and species for accurate quantification and construction of the complex and dynamic polyadenylation landscape and enables us to decode alternative polyadenylation events invisible to conventional methods at a much higher resolution.

scDAPA: detection and visualization of dynamic alternative polyadenylation from single cell RNA-seq data.

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.

Intragenic heterochromatin-mediated alternative polyadenylation modulates miRNA and pollen development in rice.

Despite a much higher proportion of intragenic heterochromatin-containing genes in crop genomes, the importance of intragenic heterochromatin in crop development remains unclear. Intragenic heterochromatin can be recognised by a protein complex, ASI1-AIPP1-EDM2 (AAE) complex, to regulate alternative polyadenylation. Here, we investigated the impact of rice ASI1 on global poly(A) site usage through poly(A) sequencing and ASI1-dependent regulation on rice development. We found that OsASI1 is essential for rice pollen development and flowering. OsASI1 dysfunction has an important impact on global poly(A) site usage, which is closely related to heterochromatin marks. Intriguingly, OsASI1 interacts with the intronic heterochromatin of OsXRNL, a nuclear XRN family exonuclease gene involved in the processing of an miRNA precursor, to promote the processing of full-length OsXRNL and regulate miRNA abundance. We found that OsASI1-mediated regulation of pollen development partially depends on OsXRNL. Finally, we characterised the rice AAE complex and its involvement in alternative polyadenylation and pollen development. Our findings help to elucidate an epigenetic mechanism governing miRNA abundance and rice development, and provide a valuable resource for studying the epigenetic mechanisms of many important processes in crops.

Alternative polyadenylated mRNAs behave as asynchronous rhythmic transcription in Arabidopsis.

Alternative polyadenylation (APA) is a widespread post-transcriptional modification method that changes the 3' ends of transcripts by altering poly(A) site usage. However, the longitudinal transcriptomic 3' end profile and its mechanism of action are poorly understood. We applied diurnal time-course poly(A) tag sequencing (PAT-seq) for Arabidopsis and identified 3284 genes that generated both rhythmic and arrhythmic transcripts. These two classes of transcripts appear to exhibit dramatic differences in expression and translation activisty. The asynchronized transcripts derived by APA are embedded with different poly(A) signals, especially for rhythmic transcripts, which contain higher AAUAAA and UGUA signal proportions. The Pol II occupancy maximum is reached upstream of rhythmic poly(A) sites, while it is present directly at arrhythmic poly(A) sites. Integrating H3K9ac and H3K4me3 time-course data analyses revealed that transcriptional activation of histone markers may be involved in the differentiation of rhythmic and arrhythmic APA transcripts. These results implicate an interplay between histone modification and RNA 3'-end processing, shedding light on the mechanism of transcription rhythm and alternative polyadenylation.

Genome-wide distribution and functions of the AAE complex in epigenetic regulation in Arabidopsis.

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context. Previous studies have shown that a protein complex, the ASI1-AIPP1-EDM2 (AAE) complex, participates in polyadenylation regulation of several intronic heterochromatin-containing genes. However, the genome-wide functions of AAE are still unknown. Here, we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome-wide level and preferentially interacts with genetic heterochromatin. Polyadenylation (poly(A) sequencing reveals that AAE complex has a substantial influence on poly(A) site usage of heterochromatin-containing genes, including not only intronic heterochromatin-containing genes but also the genes showing overlap with heterochromatin. Intriguingly, AAE is also involved in the alternative splicing regulation of a number of heterochromatin-overlapping genes, such as the disease resistance gene RPP4. We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation. In addition to conferring RNA processing regulation at genic heterochromatin-containing genes, AAE also targets some transposable elements (TEs) outside of genes (including TEs sandwiched by genes and island TEs) for epigenetic silencing. Our results reveal new functions of AAE in RNA processing and epigenetic silencing, and thus represent important advances in epigenetic regulation.

Distinct genome-wide alternative polyadenylation during the response to silicon availability in the marine diatom Thalassiosira pseudonana.

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.

Adaptive transgenerational effects remain significant.

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.

Alternative polyadenylation is involved in auxin-based plant growth and development.

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.

Transgenerational effects benefit offspring across diverse environments: a meta-analysis in plants and animals.

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.

Genome-wide dynamics of alternative polyadenylation in rice.

Alternative polyadenylation (APA), in which a transcript uses one of the poly(A) sites to define its 3'-end, is a common regulatory mechanism in eukaryotic gene expression. However, the potential of APA in determining crop agronomic traits remains elusive. This study systematically tallied poly(A) sites of 14 different rice tissues and developmental stages using the poly(A) tag sequencing (PAT-seq) approach. The results indicate significant involvement of APA in developmental and quantitative trait loci (QTL) gene expression. About 48% of all expressed genes use APA to generate transcriptomic and proteomic diversity. Some genes switch APA sites, allowing differentially expressed genes to use alternate 3' UTRs. Interestingly, APA in mature pollen is distinct where differential expression levels of a set of poly(A) factors and different distributions of APA sites are found, indicating a unique mRNA 3'-end formation regulation during gametophyte development. Equally interesting, statistical analyses showed that QTL tends to use APA for regulation of gene expression of many agronomic traits, suggesting a potential important role of APA in rice production. These results provide thus far the most comprehensive and high-resolution resource for advanced analysis of APA in crops and shed light on how APA is associated with trait formation in eukaryotes.

Genetic admixture accelerates invasion via provisioning rapid adaptive evolution.

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.

Heavy metal rich stone-processing wastewater inhibits the growth and development of plants.

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.

Role of cleavage and polyadenylation specificity factor 100: anchoring poly(A) sites and modulating transcription termination.

CPSF100 is a core component of the cleavage and polyadenylation specificity factor (CPSF) complex for 3'-end formation of mRNA, but it still has no clear functional assignment. CPSF100 was reported to play a role in RNA silencing and promote flowering in Arabidopsis. However, the molecular mechanisms underlying these phenomena are not fully understood. Our genetics analyses indicate that plants with a hypomorphic mutant of CPSF100 (esp5) show defects in embryogenesis, reduced seed production or altered root morphology. To unravel this puzzle, we employed a poly(A) tag sequencing protocol and uncovered a different poly(A) profile in esp5. This transcriptome-wide analysis revealed alternative polyadenylation of thousands of genes, most of which result in transcriptional read-through in protein-coding genes. AtCPSF100 also affects poly(A) signal recognition on the far-upstream elements; in particular it prefers less U-rich sequences. Importantly, AtCPSF100 was found to exert its functions through the change of poly(A) sites on genes encoding binding proteins, such as nucleotide-binding, RNA-binding and poly(U)-binding proteins. In addition, through its interaction with RNA Polymerase II C-terminal domain (CTD) and affecting the expression level of CTD phosphatase-like 3 (CPL3), AtCPSF100 is shown to potentially ensure transcriptional termination by dephosphorylation of Ser2 on the CTD. These data suggest a key role for CPSF100 in locating poly(A) sites and affecting transcription termination.

Genome-wide identification and predictive modeling of polyadenylation sites in eukaryotes.

Polyadenylation [poly(A)] is a vital step in post-transcriptional processing of pre-mRNA. Alternative polyadenylation is a widespread mechanism of regulating gene expression in eukaryotes. Defining poly(A) sites contributes to the annotation of transcripts' ends and the study of gene regulatory mechanisms. Here, we survey methods for collecting poly(A) sites using high-throughput sequencing technologies and summarize the general processes for genome-wide poly(A) site identifications. We also compare the performances of various poly(A) site prediction models and discuss the relationship between poly(A) site identification from sequencing projects and predictive modeling. Moreover, we attempt to address some potential problems in current researches and propose future directions related to polyadenylation research.

PASPA: a web server for mRNA poly(A) site predictions in plants and algae.

MOTIVATION: Polyadenylation is an essential process during eukaryotic gene expression. Prediction of poly(A) sites helps to define the 3' end of genes, which is important for gene annotation and elucidating gene regulation mechanisms. However, due to limited knowledge of poly(A) signals, it is still challenging to predict poly(A) sites in plants and algae. PASPA is a web server for P: oly( A: ) S: ite prediction in P: lants and A: lgae, which integrates many in-house tools as add-ons to facilitate poly(A) site prediction, visualization and mining. This server can predict poly(A) sites for ten species, including seven previously poly(A) signal non-characterized species, with sensitivity and specificity in a range between 0.80 and 0.95.

Genome-wide characterization of intergenic polyadenylation sites redefines gene spaces in Arabidopsis thaliana.

BACKGROUND: Messenger RNA polyadenylation is an essential step for the maturation of most eukaryotic mRNAs. Accurate determination of poly(A) sites helps define the 3'-ends of genes, which is important for genome annotation and gene function research. Genomic studies have revealed the presence of poly(A) sites in intergenic regions, which may be attributed to 3'-UTR extensions and novel transcript units. However, there is no systematically evaluation of intergenic poly(A) sites in plants. RESULTS: Approximately 16,000 intergenic poly(A) site clusters (IPAC) in Arabidopsis thaliana were discovered and evaluated at the whole genome level. Based on the distributions of distance from IPACs to nearby sense and antisense genes, these IPACs were classified into three categories. About 70 % of them were from previously unannotated 3'-UTR extensions to known genes, which would extend 6985 transcripts of TAIR10 genome annotation beyond their 3'-ends, with a mean extension of 134 nucleotides. 1317 IPACs were originated from novel intergenic transcripts, 37 of which were likely to be associated with protein coding transcripts. 2957 IPACs corresponded to antisense transcripts for genes on the reverse strand, which might affect 2265 protein coding genes and 39 non-protein-coding genes, including long non-coding RNA genes. The rest of IPACs could be originated from transcriptional read-through or gene mis-annotations. CONCLUSIONS: The identified IPACs corresponding to novel transcripts, 3'-UTR extensions, and antisense transcription should be incorporated into current Arabidopsis genome annotation. Comprehensive characterization of IPACs from this study provides insights of alternative polyadenylation and antisense transcription in plants.