Alternative polyadenylation (APA) is an important contributor to the regulation of gene expression in plants. One subunit of the complex that cleaves and polyadenylates mRNAs in the nucleus, CPSF30 (for the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor), has been implicated in a wide-ranging network of regulatory events. CPSF30 plays roles in root development, flowering time, and response to biotic and abiotic stresses. CPSF30 also is a conduit that links cellular signaling and RNA modification with alternative RNA processing events and transcriptional dynamics. While much is known about CPSF30 and its roles in plants, questions remain regarding the connections between CPSF30-mediated APA and the downstream events that lead to specific phenotypic outcomes. To address these, we conducted a detailed analysis of poly(A) site usage in the CPSF30 mutant. Our results corroborate earlier reports that link CPSF30 with a distinctive cis element (AAUAAA) that is present 10-30 nts upstream of some, but not all, plant pre-mRNAs. Interestingly, our results reveal a distinctive shift in poly(A) site in mutants deficient in CPSF30, resulting in cleavage and polyadenylation at the location of motifs similar to AAUAAA. Importantly, CPSF30-associated APA had at best a small impact on mRNA functionality. These results necessitate the formulation of new hypotheses for mechanisms by which CPSF30-mediated APA influences physiological processes.

PROTEIN L-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) promotes seed vigor by repairing damaged proteins. However, whether PIMT variants have arisen during maize (Zea mays) domestication remains unknown. Here, we found 2 variants in the ZmPIMT1 promoter. The ZmPIMT1  Hap C7-2 promoter exhibited stronger activity than the ZmPIMT1  Hap Z58 promoter. Maize inbred lines carrying the ZmPIMT1  Hap C7-2 promoter had greater seed vigor than ZmPIMT1  Hap Z58 lines in a population of Zhengdan 958 recombinant inbred lines (RILs) and a maize inbred population. By characterizing the maize zmpimt1 knockdown mutant, ZmPIMT1-overexpressing maize and Arabidopsis thaliana heterologous ZmPIMT1 overexpression lines, we demonstrated that ZmPIMT1 positively regulates seed vigor. Co-IP and LC-MS/MS assays showed that ZmPIMT1 interacts with and repairs damaged POLY(A) BINDING PROTEIN2 (PABP2). ZmPIMT1 stabilizes PABP2 RNA-binding activity and regulates the stability and translation efficiency of the mRNA during maize seed germination. Disruption of PABP2 decreases seed vigor in Arabidopsis thaliana. Furthermore, the F-statistics (Fixation index; FST) and nucleotide diversity (θπ) ratio between teosinte and maize lines showed that ZmPIMT1 likely has not undergone selection during maize domestication. Our findings unveil a molecular mechanism in which ZmPIMT1 regulates seed vigor in maize and highlight a potential application of the advantageous ZmPIMT1 haplotype for breeding new varieties with increased seed vigor.

Genomics is an increasingly important part of biology research. However, educating undergraduates in genomics is not yet a standard part of life sciences curricula. We believe this is, in part, due to a lack of standard concepts for the teaching of genomics. To address this deficit, the members of the Genomics Education Alliance created a set of genomics concepts that was then further refined by input from a community of undergraduate educators who engage in genomics instruction. The final genomics concepts list was compared to existing learning frameworks, including the Vision and Change initiative (V&C), as well as ones for genetics, biochemistry and molecular biology, and bioinformatics. Our results demonstrate that the new genomics framework fills a niche not addressed by previous inventories. This new framework should be useful to educators seeking to design stand-alone courses in genomics as well as those seeking to incorporate genomics into existing coursework. Future work will involve designing curriculum and assessments to go along with this genomics learning framework.

Plasmodium falciparum is an obligate human parasite of the phylum Apicomplexa and is the causative agent of the most lethal form of human malaria. Although N6-methyladenosine modification is thought to be one of the major post-transcriptional regulatory mechanisms for stage-specific gene expression in apicomplexan parasites, the precise base position of m6A in mRNAs or noncoding RNAs in these parasites remains unknown. Here, we report global nucleotide-resolution mapping of m6A residues in P. falciparum using DART-seq technology, which quantitatively displayed a stage-specific, dynamic distribution pattern with enrichment near mRNA 3' ends. In this process we identified 894, 788, and 1,762 m6A-modified genes in Ring, Trophozoite and Schizont stages respectively, with an average of 5-7 m6A sites per-transcript at the individual gene level. Notably, several genes involved in malaria pathophysiology, such as KAHRP, ETRAMPs, SERA and stress response genes, such as members of Heat Shock Protein (HSP) family are highly enriched in m6A and therefore could be regulated by this RNA modification. Since we observed preferential methylation at the 3' ends of P. falciparum transcripts and because malaria polyadenylation specificity factor PfCPSF30 harbors an m6A reader 'YTH' domain, we reasoned that m6A might play an important role in 3'-end processing of malaria mRNAs. To investigate this, we used two complementary high-throughput RNA 3'-end mapping approaches, which provided an initial framework to explore potential roles of m6A in the regulation of alternative polyadenylation (APA) during malaria development in human hosts.

In this study, we investigated the interplay between the spermosphere inoculum, host plant physiology, and endophytic compartment (EC) microbial community. Using 16S ribosomal RNA gene sequencing of root, stem, and leaf endophytic compartment communities, we established a baseline microbiome for Nicotiana sp. Phenotypic differences were observed due to the addition of some bacterial inoculants, correlated with endogenous auxin loads using transgenic plants expressing the auxin reporter pB-GFP::P87. When applied as spermosphere inoculants, select bacteria were found to create reproducible variation within the root EC microbiome and, more systematically, the host plant physiology. Our findings support the assertion that the spermosphere of plants is a zone that can influence the EC microbiome when applied in a greenhouse setting.

Messenger RNA polyadenylation, the process wherein the primary RNA polymerase II transcript is cleaved and a poly(A) tract added, is a key step in the expression of genes in plants. Moreover, it is a point at which gene expression may be regulated by determining the functionality of the mature mRNA. Polyadenylation is mediated by a complex (the polyadenylation complex, or PAC) that consists of between 15 and 20 subunits. While the general functioning of these subunits may be inferred by extending paradigms established in well-developed eukaryotic models, much remains to be learned about the roles of individual subunits in the regulation of polyadenylation in plants. To gain further insight into this, we conducted a survey of variability in the plant PAC. For this, we drew upon a database of naturally-occurring variation in numerous geographic isolates of Arabidopsis thaliana. For a subset of genes encoding PAC subunits, the patterns of variability included the occurrence of premature stop codons in some Arabidopsis accessions. These and other observations lead us to conclude that some genes purported to encode PAC subunits in Arabidopsis are actually pseudogenes, and that others may encode proteins with dispensable functions in the plant. Many subunits of the PAC showed patterns of variability that were consistent with their roles as essential proteins in the cell. Several other PAC subunits exhibit patterns of variability consistent with selection for new or altered function. We propose that these latter subunits participate in regulatory interactions important for differential usage of poly(A) sites.

The late embryogenesis abundant proteins (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs’ presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thaliana seed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high-throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire subpopulation of phage retained by each LEAP ortholog. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (paired-end phage sequencing) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two dehydrin orthologs (LEA14 and GmPm12) compared to bovine serum albumin (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature-related intensity change analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation assays. The results provide insights into the involvement of the dehydrin Y-segments and K-domains in protein binding.

Systemic acquired resistance (SAR) involves the generation of systemically transported signal that arms distal plant parts against secondary infections. We show that two phased 21-nucleotide (nt) trans-acting small interfering RNA3a RNAs (tasi-RNA) derived from TAS3a and synthesized within 3 hours of pathogen infection are the early mobile signal in SAR. TAS3a undergoes alternate polyadenylation, resulting in the generation of 555- and 367-nt transcripts. The 555-nt transcripts likely serves as the sole precursor for tasi-RNAs D7 and D8, which cleave Auxin response factors (ARF) 2, 3, and 4 to induce SAR. Conversely, increased expression of ARF3 represses SAR. Knockout mutations in TAS3a or RNA silencing components required for tasi-RNA biogenesis compromise SAR without altering levels of known SAR-inducing chemicals. Both tasi-ARFs and the 367-nt transcripts are mobile and transported via plasmodesmata. Together, we show that tasi-ARFs are the early mobile signal in SAR.

Recent years have seen an explosion of interest in the subject of alternative polyadenylation in plants. Connections between the polyadenylation complex and numerous developmental and stress responses are well-established. However, those that link stimuli with the functioning of the polyadenylation complex are less well understood. To this end, it is imperative to clearly delineate the roles of the polyadenylation complex in both plant growth AND alternative polyadenylation. It is also necessary to understand the ways by which other molecular processes may contribute to alternative polyadenylation. This review discusses these issues, with a focus on instances that reveal mechanisms by which mRNA polyadenylation may be regulated. Insights from from characterizations of mutants affected in the polyadenylation complex are discussed, as are the limitations of such characterizations when it comes to teasing out cause and effect. These limitations encourage explorations to other processes that are beyond the core polyadenylation complex. Two such processes that sculpt the plant transcriptome - transcription termination and the epigenetic control of transposon activity - also contribute to regulated poly(A) site choice. These subjects define "the right places" - molecular mechanisms that contribute to the wide-ranging control of gene expression via mRNA polyadenylation.

Alternative polyadenylation—the series of events that leads to the production of different mRNA isoforms from a primary transcript via usage of different polyadenylation sites within the transcript-is an important facet of gene expression in plants.Among the pro-teins that control alternative polyadenylation in plants is CPSF30,a core subunit of the polyadenylation complex(Chakrabarti and Hunt,2015).In plants,CPSF30 integrates cellular signaling with numerous growth and developmental processes through an interaction with calmodulin(Chakrabarti and Hunt,2015).

In recent years, a class of chemical compounds (benzoxaboroles) that are active against a range of parasites has been shown to target mRNA polyadenylation by inhibiting the activity of CPSF73, the endonucleolytic core of the eukaryotic polyadenylation complex. One particular compound, termed AN3661, is active against several apicomplexan parasites that cause disease in humans. In this study, we report that AN3661 is active against an apicomplexan that causes disease in horses and marine mammals ( Sarcocystis neurona ), with an approximate IC 50 value of 14.99 nM. Consistent with the reported mode of action of AN3661 against other apicomplexans, S . neurona mutants resistant to AN3661 had an alteration in CPSF73 that was identical to a mutation previously documented in AN3661-resistant Toxoplasma gondii and Plasmodium falciparum . AN3661 had a wide-ranging effect on poly(A) site choice in S . neurona , with more than half of all expressed genes showing some alteration in mRNA 3’ ends. This was accompanied by changes in the relative expression of more than 25% of S . neurona genes and an overall 5-fold reduction of S . neurona transcripts in infected cells. In contrast, AN3661 had no discernible effect on poly(A) site choice or gene expression in the host cells. These transcriptomic studies indicate that AN3661 is exceedingly specific for the parasite CPSF73 protein, and has the potential to augment other therapies for the control of apicomplexan parasites in domestic animals.

Drought tolerance is a crucial trait for crops to curtail the yield loss inflicted by water stress, yet genetic improvement efforts are challenged by the complexity of this character. The adaptation of sorghum to abiotic stress, its genotypic variability, and relatively small genome make this species well-suited to dissect the molecular basis of drought tolerance. The use of differential transcriptome analysis provides a snapshot of the bioprocesses underlying drought response as well as genes that might be determinants of the drought tolerance trait. RNA sequencing data were analyzed via gene ontology enrichment to compare the transcriptome profiles of two sorghum lines, the drought-tolerant SC56 and the drought-sensitive Tx7000. SC56 outperformed Tx7000 in wet conditions by upregulating processes driving growth and guaranteeing homeostasis. The drought tolerance of SC56 seems to be an intrinsic trait occurring through overexpressing stress tolerance genes in wet conditions, notably genes acting in defense against oxidative stress (SOD1, SOD2, VTC1, MDAR1, MSRB2, and ABC1K1). Similarly to wet conditions, under drought, SC56 enhanced its transmembrane transport and maintained growth-promoting mechanisms. Under drought, SC56 also upregulated stress tolerance genes that heighten the antioxidant capacity (SOD1, RCI3, VTE1, UCP1, FD1, and FD2), regulatory factors (CIPK1 and CRK7), and repressors of premature senescence (SAUL1). The differential expression analysis uncovered biological processes which upregulation enables SC56 to be a better accumulator of biomass and connects the drought tolerance trait to key stress tolerance genes, making this genotype a judicious choice for isolation of tolerance genes.

Alternative polyadenylation (APA) regulates diverse developmental and physiological processes through its effects on gene expression, mRNA stability, translatability, and transport. Sorghum is a major cereal crop in the world and, despite its importance, not much is known about the role of post-transcriptional regulation in mediating responses to abiotic stresses in Sorghum. A genome-wide APA analysis unveiled widespread occurrence of APA in Sorghum in response to drought, heat, and salt stress. Abiotic stress treatments incited changes in poly(A) site choice in a large number of genes. Interestingly, abiotic stresses led to the re-directing of transcriptional output into non-productive pathways defined by the class of poly(A) site utilized. This result revealed APA to be part of a larger global response of Sorghum to abiotic stresses that involves the re-direction of transcriptional output into non-productive transcriptional and translational pathways. Large numbers of stress-inducible poly(A) sites could not be linked with known, annotated genes, suggestive of the existence of numerous unidentified genes whose expression is strongly regulated by abiotic stresses. Furthermore, we uncovered a novel stress-specific cis-element in intronic poly(A) sites used in drought- and heat-stressed plants that might play an important role in non-canonical poly(A) site choice in response to abiotic stresses.

Nitrogen (N) is probably the most important macronutrient and its scarcity limits plant growth, development and fitness. N starvation response has been largely studied by transcriptomic analyses, but little is known about the role of alternative polyadenylation (APA) in such response. In this work, we show that N starvation modifies poly(A) usage in a large number of transcripts, some of them mediated by FIP1, a component of the polyadenylation machinery. Interestingly, the number of mRNAs isoforms with poly(A) tags located in protein-coding regions or 5′-UTRs significantly increases in response to N starvation. The set of genes affected by APA in response to N deficiency is enriched in N-metabolism, oxidation-reduction processes, response to stresses, and hormone responses, among others. A hormone profile analysis shows that the levels of salicylic acid (SA), a phytohormone that reduces nitrate accumulation and root growth, increase significantly upon N starvation. Meta-analyses of APA-affected and fip1-2-deregulated genes indicate a connection between the nitrogen starvation response and salicylic acid (SA) signaling. Genetic analyses show that SA may be important for preventing the overgrowth of the root system in low N environments. This work provides new insights on how plants interconnect different pathways, such as defense-related hormonal signaling and the regulation of genomic information by APA, to fine-tune the response to low N availability.

3' end processing and mRNA polyadenylation is a vital aspect of gene expression in eukaryotes, and also a step at which expression may be regulated. A spate of recent research in plants links different subunits of the polyadenylation complex with growth and development. These reports provide insight into mechanisms by which APA may be regulated, and perhaps into mechanisms by which pre-mRNAs are processed and polyadenylated. In this review, several of these recent reports are discussed, with the purpose of highlighting novel features of mRNA 3' end formation in plants and also developing broad themes that connect APA with plant growth, development, and responses to the environment. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Development.

Among the Lamiaceae family, the genus Thymus is an economically important genera due to its medicinal and aromatic properties. Most Thymus molecular research has focused on the determining the phylogenetic relationships between different species, but no published work has focused on the evolution of the transcriptome across the genus to elucidate genes involved in terpenoid biosynthesis. Hence, in this study, the transcriptomes of five different Thymus species were generated and analyzed to mine putative genes involved in thymol and carvacrol biosynthesis. High-throughput sequencing produced ~43 million high-quality reads per sample, which were assembled de novo using several tools, then further subjected to a quality evaluation. The best assembly for each species was used as queries to search within the UniProt, KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups) and TF (Transcription Factors) databases. Mining the transcriptomes resulted in the identification of 592 single-copy orthogroups used for phylogenetic analysis. The data showed strongly support a close genetic relationship between Thymus vulgaris and Thymus daenensis. Additionally, this study dates the speciation events between 1.5-2.1 and 9-10.2 MYA according to different methodologies. Our study provides a global overview of genes related to the terpenoid pathway in Thymus, and can help establish an understanding of the relationship that exists among Thymus species.

Root development and its response to environmental changes is crucial for whole plant adaptation. These responses include changes in transcript levels. Here, we show that the alternative polyadenylation (APA) of mRNA is important for root development and responses. Mutations in FIP1, a component of polyadenylation machinery, affects plant development, cell division and elongation, and response to different abiotic stresses. Salt treatment increases the amount of poly(A) site usage within the coding region and 5' untranslated regions (5'-UTRs), and the lack of FIP1 activity reduces the poly(A) site usage within these non-canonical sites. Gene ontology analyses of transcripts displaying APA in response to salt show an enrichment in ABA signaling, and in the response to stresses such as salt or cadmium (Cd), among others. Root growth assays show that fip1-2 is more tolerant to salt but is hypersensitive to ABA or Cd. Our data indicate that FIP1-mediated alternative polyadenylation is important for plant development and stress responses.

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.

Table S5. Bioprocesses and pathways upregulated in Nosema-infected bees under poor diet. Upregulated bioprocesses (GO-terms) and Kegg pathways in Nosema-infected bees under poor diet feeding (carbohydrates-only) compared to pollen (C > P), Bee-Pro (C > B) and MegaBee (CN > MN). (XLSX 92 kb)

Messenger RNA polyadenylation is a universal aspect of gene expression in eukaryotes. In well-established model organisms, this process is mediated by a conserved complex of 15-20 subunits. To better understand this process in apicomplexans, a group of unicellular parasites that causes serious disease in humans and livestock, a computational and high throughput sequencing study of the polyadenylation complex and poly(A) sites in several species was conducted. BLAST-based searches for orthologs of the human polyadenylation complex yielded clear matches to only two-poly(A) polymerase and CPSF73-of the 19 proteins used as queries in this analysis. As the human subunits that recognize the AAUAAA polyadenylation signal (PAS) were not immediately obvious, a computational analysis of sequences adjacent to experimentally-determined apicomplexan poly(A) sites was conducted. The results of this study showed that there exists in apicomplexans an A-rich region that corresponds in position to the AAUAAA PAS. The set of experimentally-determined sites in one species, Sarcocystis neurona, was further analyzed to evaluate the extent and significance of alternative poly(A) site choice in this organism. The results showed that almost 80% of S. neurona genes possess more than one poly(A) site, and that more than 780 sites showed differential usage in the two developmental stages-extracellular merozoites and intracellular schizonts-studied. These sites affected more than 450 genes, and included a disproportionate number of genes that encode membrane transporters and ribosomal proteins. Taken together, these results reveal that apicomplexan species seem to possess a poly(A) signal analogous to AAUAAA even though genes that may encode obvious counterparts of the AAUAAA-recognizing proteins are absent in these organisms. They also indicate that, as is the case in other eukaryotes, alternative polyadenylation is a widespread phenomenon in S. neurona that has the potential to impact growth and development.