RNA Regulatory Elements and Polyadenylation in Plants

Alternative poly(A) site choice (also known as alternative polyadenylation, or APA) has the potential to affect gene expression in qualitative and quantitative ways. APA may affect as many as 82% of all expressed genes in a plant. The consequences of APA include the generation of transcripts with differing 3′-UTRs (and thus differing regulatory potential) and of transcripts with differing protein-coding potential. Genome-wide studies of possible APA suggest a linkage with pre-mRNA splicing, and indicate a coincidence of and perhaps cooperation between RNA regulatory elements that affect splicing efficiency and the recognition of novel intronic poly(A) sites. These studies also raise the possibility of the existence of a novel class of polyadenylation-related cis elements that are distinct from the well-characterized plant polyadenylation signal. Many potential APA events, however, have not been associated with identifiable cis elements. The present state of the field reveals a broad scope of APA, and also numerous opportunities for research into mechanisms that govern both choice and regulation of poly(A) sites in plants

CPSF30 at the Interface of Alternative Polyadenylation and Cellular Signaling in Plants

Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3\u27 end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules

Ribonuclease activity is a common property of Arabidopsis CCCH-containing zinc-finger proteins

AbstractThe CCCH class of zinc fingers occurs in a large number of Arabidopsis proteins. Previous studies revealed that one such protein is a nuclease, the activity of which is attributable to one of the CCCH motifs. To examine whether nuclease activity is a more general characteristic of CCCH zinc finger containing proteins, five other such Arabidopsis proteins were assayed for a similar activity. The results indicate that all of these proteins possess nuclease activity. Thus, nuclease activity may be a common characteristic of Arabidopsis CCCH-containing proteins

A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3′ ends with hydroxyl groups, as they can be labeled by RNA ligase with [32P]-cytidine-3′,5′-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1

A Novel Endonuclease Activity Associated with the \u3cem\u3eArabidopsis\u3c/em\u3e Ortholog of the 30-kDa Subunit of Cleavage and Polyadenylation Specificity Factor

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3′ ends with hydroxyl groups, as they can be labeled by RNA ligase with [32P]-cytidine-3′,5′-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1

Use of HRMA Proteins and Their Genes for Broad Range Protection of Plants Against Bacterial, Fungal and Viral Pathogens

The use of an avr gene hrmA to induce systematic acquired resistance in plant cells, plant seeds, plant tissues and plants is disclosed. Also disclosed is the use of low level expression of promoters in combination with the hrmA gene to provide broad-spectrum pathogen resistance in plant cells, plant seeds, plant tissues and plants

Genome-Wide Atlas of Alternative Polyadenylation in the Forage Legume Red Clover

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

Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

BACKGROUND: 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. RESULTS: It was found that AtCPSF30 by itself localizes, not to the nucleus, but to the cytoplasm. AtCPSF30 could be found in the nucleus when co-expressed with AtCPSF160 or AtCPSF73(I), one of the two Arabidopsis orthologs of CPSF73. This re-directing of AtCPSF30 indicates that AtCPSF30 is retained in the nucleus via interactions with either or both of these other CPSF subunits. Co-expression of AtCSPF30 with AtCPSF100 altered the location, not of AtCPSF30, but rather of AtCPSF100, with these proteins residing in the cytoplasm. Deletion of plant-specific N- or C-terminal domains of AtCPSF30 abolished various of the interactions between AtCPSF30 and other CPSF subunits, suggesting that the plant CPSF complex assembles via novel protein-protein interactions. CONCLUSION: These results suggest that the nuclear CPSF complex in plants is a dynamic one, and that the interactions between AtCPSF30 and other CPSF subunits are different from those existing in other eukaryotes

Plant polyadenylation factors: conservation and variety in the polyadenylation complex in plants

BACKGROUND: 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. RESULTS: The evolutionary trajectories revealed several interesting features of plant polyadenylation factors. First, the number of genes encoding plant polyadenylation factors was clearly increased from lower to higher plants. Second, the gene expansion in higher plants was biased to some polyadenylation factors, particularly those involved in RNA binding. Finally, while there are clear commonalities, the differences in the polyadenylation apparatus were obvious across different species, suggesting an ongoing process of evolutionary change. These features lead to a model in which the plant polyadenylation complex consists of a conserved core, which is rather rigid in terms of evolutionary conservation, and a panoply of peripheral subunits, which are less conserved and associated with the core in various combinations, forming a collection of somewhat distinct complex assemblies. CONCLUSIONS: The multiple forms of plant polyadenylation complex, together with the diversified polyA signals may explain the intensive alternative polyadenylation (APA) and its regulatory role in biological functions of higher plants

Characterization of mRNA Polyadenylation in the Apicomplexa

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