Publications

Polyadenylation factor CLP1 is essential for mRNA 3'-end processing in yeast and mammals. The Arabidopsis (Arabidopsis thaliana) CLP1-SIMILAR PROTEIN3 (CLPS3) is an ortholog of human hCLP1. CLPS3 was previously found to be a subunit in the affinity-purified PCFS4-TAP (tandem affinity purification) complex involved in the alternative polyadenylation of FCA and flowering time control in Arabidopsis. In this article, we further explored the components in the affinity-purified CLPS3-TAP complex, from which Arabidopsis cleavage and polyadenylation specificity factor (CPSF) subunits AtCPSF100 and AtCPSF160 were found. This result implies that CLPS3 may bridge CPSF to the PCFS4 complex. Characterization of the CLPS3 mutant revealed that CLPS3 was essential for embryo development and important for female gametophyte transmission. Overexpression of CLPS3-TAP fusion caused a range of postembryonic development abnormalities, including early flowering time, altered phyllotaxy, and abnormal numbers and shapes of flower organs. These phenotypes are associated with the altered gene expression levels of FCA, WUS, and CUC1. The decreased ratio of FCA-beta to FCA-gamma in the overexpression plants suggests that CLPS3 favored the usage of FCA regular poly(A) site over the alternative site. These observations indicate that Arabidopsis CLPS3 might be involved in the processing of pre-mRNAs encoded by a distinct subset of genes that are important in plant development.

BACKGROUND: Plants respond to many unfavorable environmental conditions via signaling mediated by altered levels of various reactive oxygen species (ROS). To gain additional insight into oxidative signaling responses, Arabidopsis mutants that exhibited tolerance to oxidative stress were isolated. We describe herein the isolation and characterization of one such mutant, oxt6.

METHODOLOGY/PRINCIPAL FINDINGS: The oxt6 mutation is due to the disruption of a complex gene (At1g30460) that encodes the Arabidopsis ortholog of the 30-kD subunit of the cleavage and polyadenylation specificity factor (CPSF30) as well as a larger, related 65-kD protein. Expression of mRNAs encoding Arabidopsis CPSF30 alone was able to restore wild-type growth and stress susceptibility to the oxt6 mutant. Transcriptional profiling and single gene expression studies show elevated constitutive expression of a subset of genes that encode proteins containing thioredoxin- and glutaredoxin-related domains in the oxt6 mutant, suggesting that stress can be ameliorated by these gene classes. Bulk poly(A) tail length was not seemingly affected in the oxt6 mutant, but poly(A) site selection was different, indicating a subtle effect on polyadenylation in the mutant.

CONCLUSIONS/SIGNIFICANCE: These results implicate the Arabidopsis CPSF30 protein in the posttranscriptional control of the responses of plants to stress, and in particular to the expression of a set of genes that suffices to confer tolerance to oxidative stress.

BACKGROUND: One of the essential processing events during pre-mRNA maturation is the post-transcriptional addition of a polyadenine [poly(A)] tail. The 3'-end poly(A) track protects mRNA from unregulated degradation, and indicates the integrity of mRNA through recognition by mRNA export and translation machinery. The position of a poly(A) site is predetermined by signals in the pre-mRNA sequence that are recognized by a complex of polyadenylation factors. These signals are generally tri-part sequence patterns around the cleavage site that serves as the future poly(A) site. In plants, there is little sequence conservation among these signal elements, which makes it difficult to develop an accurate algorithm to predict the poly(A) site of a given gene. We attempted to solve this problem.

RESULTS: Based on our current working model and the profile of nucleotide sequence distribution of the poly(A) signals and around poly(A) sites in Arabidopsis, we have devised a Generalized Hidden Markov Model based algorithm to predict potential poly(A) sites. The high specificity and sensitivity of the algorithm were demonstrated by testing several datasets, and at the best combinations, both reach 97%. The accuracy of the program, called poly(A) site sleuth or PASS, has been demonstrated by the prediction of many validated poly(A) sites. PASS also predicted the changes of poly(A) site efficiency in poly(A) signal mutants that were constructed and characterized by traditional genetic experiments. The efficacy of PASS was demonstrated by predicting poly(A) sites within long genomic sequences.

CONCLUSION: Based on the features of plant poly(A) signals, a computational model was built to effectively predict the poly(A) sites in Arabidopsis genes. The algorithm will be useful in gene annotation because a poly(A) site signifies the end of the transcript. This algorithm can also be used to predict alternative poly(A) sites in known genes, and will be useful in the design of transgenes for crop genetic engineering by predicting and eliminating undesirable poly(A) sites.

The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.

It has been reported that the expression of the yeast poly(A) binding protein gene (PAB1) in plants leads to an induction of disease resistance responses, accompanied by alterations in the growth habit of the plant (Li et al. Plant Mol. Biol. (2000) 42 335). To capitalize on this observation, a feedback-regulated PAB1 gene was assembled and introduced into tobacco and Arabidopsis. The regulation entailed the linking of the expression of the PAB1 gene to control by the lac repressor, and by linking lac repressor expression to the disease resistance state of the plant, such that the induction of systemic defense responses by accumulation of the yeast poly(A) binding protein would turn off the expression of the PAB1 gene. Plants containing this system showed elevated and/or constitutive expression of disease-associated genes and significant resistance to otherwise pathogenic organisms. As well, they displayed a nearly normal growth habit under laboratory and greenhouse settings. These studies indicate that the expression of cytotoxic genes (such as the PAB1 gene) in plants can be controlled so that enhanced disease resistance can be achieved without significantly affecting plant growth and development.

Reverse peptide of indolicidin (Rev4), a 13-residue peptide based on the sequence of indolicidin, has been shown to possess both strong antimicrobial and protease inhibitory activities in vitro. To evaluate its efficacy in vivo, we produced and evaluated transgenic tobacco (Nicotiana tabacum L.) and Arabidopsis thaliana [(L.) Heynh.] plants expressing Rev4 with different signal peptide sequences for pathogen resistance. All transgenic plants showed normal growth and development, an indication of no or low cytotoxicity of the peptide. Furthermore, the transgenic plants exhibited elevated resistance to three bacterial and two oomycete pathogens. Interestingly, tobacco plants expressing Rev4 displayed enhanced yield compared to the control as indicated by an increased biomass production by as much as 34% in two field trials. When Rev4 was coexpressed with another antimicrobial peptide, Myp30, the disease resistance levels in the transgenic Arabidopsis were enhanced. These findings suggest the potential of using these peptides to protect plants from microbial pathogens and to enhance yield.

The cleavage and polyadenylation specificity factor (CPSF) is an important multi-subunit component of the mRNA 3'-end processing apparatus in eukaryotes. The Arabidopsis genome contains five genes encoding CPSF homologues (AtCPSF160, AtCPSF100, AtCPSF73-I, AtCPSF73-II and AtCPSF30). These CPSF homologues interact with each other in a way that is analogous to the mammalian CPSF complex or their yeast counterparts, and also interact with the Arabidopsis poly(A) polymerase (PAP). There are two CPSF73 like proteins (AtCPSF73-I and AtCPSF73-II) that share homology with the 73 kD subunit of the mammalian CPSF complex. AtCPSF73-I appears to correspond to the functionally characterized mammalian CPSF73 and its yeast counterpart. AtCPSF73-II was identified as a novel protein with uncharacterized protein homologues in other multicellular organisms, but not in yeast. Both of the AtCPSF73 proteins are targeted in the nucleus and were found to interact with AtCPSF100. They are also essential since knockout or knockdown mutants are lethal. In addition, the expression level of AtCPSF73-I is critical for Arabidopsis development because overexpression of AtCPSF73-I is lethal. Interestingly, transgenic plants carrying an additional copy of the AtCPSF73-I gene, that is, the full-length cDNA under the control of its native promoter, appeared normal but were male sterile due to delayed anther dehiscence. In contrast, we previously demonstrated that a mutation in the AtCPSF73-II gene was detrimental to the genetic transmission of female gametes. Thus, two 73 kD subunits of the AtCPSF complex appear to have special functions during flower development. The important roles of mRNA 3'-end processing machinery in modulating plant development are discussed.

Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3'-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3'-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a single-nucleotide profile scan on the 3'-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3'-ends for gene annotation.

We have identified and genetically characterized an Arabidopsis thaliana gene encoding a homolog of the Cleavage and Polyadenylation Specificity Factor (CPSF). This gene, named AtCPSF73-II, has been found to have a critical role in development by loss-of-function analysis using a Dissociation (Ds) insertion line SGT1922. The homozygous SGT1922 plants were lethal, but the heterozygous plants, while retaining their normal vegetative growth, displayed empty seed spaces as well as aborted seeds with embryos arrested at the globular stage. Genetic analysis indicated that the disruption of the AtCPSF73-II gene in SGT1922 plants caused severe reduction in genetic transmission of female gametes due to a loss of fertility, while the transmission of male gametes was normal. Two independent heterozygous lines with T-DNA insertion on the AtCPSF73-II gene also showed the similar phenotype. Gene expression analysis demonstrated that AtCPSF73-II was preferentially expressed in flowers. Protein sequence analysis revealed a group of AtCPSF73-II homologs with unknown function in animals, but not in yeast, which suggested a potential important function of this group of genes in the development of multicellular organisms.

Helper component-protease (HC-Pro) is a plant viral suppressor of RNA silencing, and transgenic tobacco expressing HC-Pro has increased susceptibility to a broad range of viral pathogens. Here we report that these plants also exhibit enhanced resistance to unrelated heterologous pathogens. Tobacco mosaic virus (TMV) infection of HC-Pro-expressing plants carrying the N resistance gene results in fewer and smaller lesions compared to controls without HC-Pro. The resistance to TMV is compromised but not eliminated by expression of nahG, which prevents accumulation of salicylic acid (SA), an important defense signaling molecule. HC-Pro-expressing plants are also more resistant to tomato black ring nepovirus (TBRV) and to the oomycete Peronospora tabacina. Enhanced TBRV resistance is SA-independent, whereas the response to P. tabacina is associated with early induction of markers characteristic of SA-dependent defense. Thus, a plant viral suppressor of RNA silencing enhances resistance to multiple pathogens via both SA-dependent and SA-independent mechanisms.