Polyadenylation, an essential step in eukaryotic gene expression, requires both cis-elements and a plethora of trans-acting polyadenylation factors. The polyadenylation factors are largely conserved across mammals and fungi. The conservation seems also extended to plants based on the analyses of Arabidopsis polyadenylation factors. To extend this observation, we systemically identified the orthologs of yeast and human polyadenylation factors from 10 plant species chosen based on both the availability of their genome sequences and their positions in the evolutionary tree, which render them representatives of different plant lineages.

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.

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.

The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to further explore the functions of AtCPSF30, the subcellular distribution of the protein was examined by over-expressing fusion proteins containing fluorescent reporters linked to different CPSF subunits.

The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using low-throughput (gene-by-gene) and high-throughput (transcriptome sequencing) approaches that recovered poly(A)-containing sequence tags which revealed interesting features of this critical process in Chlamydomonas. In this study, RNA polyadenylation has been studied using the so-called Poly(A) Tag Sequencing (PAT-Seq) approach. Specifically, PAT-Seq was used to study poly(A) site choice in cultures grown in four different media types-Tris-Phosphate (TP), Tris-Phosphate-Acetate (TAP), High-Salt (HS), and High-Salt-Acetate (HAS). The results indicate that: 1. As reported before, the motif UGUAA is the primary, and perhaps sole, cis-element that guides mRNA polyadenylation in the nucleus; 2. The scope of alternative polyadenylation events with the potential to change the coding sequences of mRNAs is limited; 3. Changes in poly(A) site choice in cultures grown in the different media types are very few in number and do not affect protein-coding potential; 4. Organellar polyadenylation is considerable and affects primarily ribosomal RNAs in the chloroplast and mitochondria; and 5. Organellar RNA polyadenylation is a dynamic process that is affected by the different media types used for cell growth.

The ability to integrate environmental and developmental signals with physiological responses is critical for plant survival. How this integration is done, particularly through posttranscriptional control of gene expression, is poorly understood. Previously, it was found that the 30 kD subunit of Arabidopsis cleavage and polyadenylation specificity factor (AtCPSF30) is a calmodulin-regulated RNA-binding protein. Here we demonstrated that mutant plants (oxt6) deficient in AtCPSF30 possess a novel range of phenotypes--reduced fertility, reduced lateral root formation, and altered sensitivities to oxidative stress and a number of plant hormones (auxin, cytokinin, gibberellic acid, and ACC). While the wild-type AtCPSF30 (C30G) was able to restore normal growth and responses, a mutant AtCPSF30 protein incapable of interacting with calmodulin (C30GM) could only restore wild-type fertility and responses to oxidative stress and ACC. Thus, the interaction with calmodulin is important for part of AtCPSF30 functions in the plant. Global poly(A) site analysis showed that the C30G and C30GM proteins can restore wild-type poly(A) site choice to the oxt6 mutant. Genes associated with hormone metabolism and auxin responses are also affected by the oxt6 mutation. Moreover, 19 genes that are linked with calmodulin-dependent CPSF30 functions, were identified through genome-wide expression analysis. These data, in conjunction with previous results from the analysis of the oxt6 mutant, indicate that the polyadenylation factor AtCPSF30 is a regulatory hub where different signaling cues are transduced, presumably via differential mRNA 3' end formation or alternative polyadenylation, into specified phenotypic outcomes. Our results suggest a novel function of a polyadenylation factor in environmental and developmental signal integration.

Studies on prevalence and significance of alternative polyadenylation (APA) in plants have been so far limited mostly to the model plants. Here, a genome-wide analysis of APA was carried out in different tissue types in the non-model forage legume red clover (Trifolium pratense L). A profile of poly(A) sites in different tissue types was generated using so-called 'poly(A)-tag sequencing' (PATseq) approach. Our analysis revealed tissue-wise dynamics of usage of poly(A) sites located at different genomic locations. We also identified poly(A) sites and underlying genes displaying APA in different tissues. Functional categories enriched in groups of genes manifesting APA between tissue types were determined. Analysis of spatial expression of genes encoding different poly(A) factors showed significant differential expression of genes encoding orthologs of FIP1(V) and PCFS4, suggesting that these two factors may play a role in regulating spatial APA in red clover. Our analysis also revealed a high degree of conservation in diverse plant species of APA events in mRNAs encoding two key polyadenylation factors, CPSF30 and FIP1(V). Together with our previously reported study of spatial gene expression in red clover, this study will provide a comprehensive account of transcriptome dynamics in this non-model forage legume.

The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A) tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A) tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis.

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.

The polyadenylation of mRNA in eukaryotes is an important biological process. In recent years, significant progress has been made in the field of mRNA polyadenylation owing to the advent of the next generation DNA sequencing technologies. The high-throughput sequencing capabilities have resulted in the direct experimental determinations of large numbers of polyadenylation sites, analysis of which has revealed a vast potential for the regulation of gene expression in eukaryotes. These collections have been generated using specialized sequencing methods that are targeted to the junction of 3'-UTR and the poly(A) tail. Here we present three variations of such a protocol that has been used for the analysis of alternative polyadenylation in plants. While all these methods use oligo-dT as an anchor to the 3'-end, they differ in the means of generating an anchor for the 5'-end in order to produce PCR products suitable for effective Illumina sequencing; the use of different methods to append 5' adapters expands the possible utility of these approaches. These methods are versatile, reproducible, and may be used for gene expression analysis as well as global determinations of poly(A) site choice.

Alternative polyadenylation (APA) plays an important role in the post-transcriptional regulation of gene expression. Little is known about how APA sites may evolve in homologous genes in different plant species. To this end, comparative studies of APA sites in different organisms are needed. In this study, a collection of poly(A) sites in Medicago truncatula, a model system for legume plants, has been generated and compared with APA sites in Arabidopsis thaliana.

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.