MESSENGER RNA 3 END FORMATION IN PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020849
• Project Start Date: Oct 10, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Plant and Soil Sciences

Non Technical Summary
Messenger RNA polyadenylation is a central step in the expression of genes in plants. In particular, mRNA polyadenylation directly impacts features that are fundamentally important for crop production and productivity - this includes the timing and success of flowering and the ability of plants to withstand environmental stresses and diseases. The research proposed herein addresses the second of these topics. Environmental stresses (heat, cold, drought, salt, etc.) and diseases pose substantial challenges for crop production; while many tools exist to help producers overcome these challenges, the dynamic nature of farming can often render these tools obsolete. For example, farms may be serially subjected to periods of cold, flooding, heat, and drought, all within one or a few growing seasons. Tools developed to address one of these insults are usually not effective against others. Likewise, while the introduction of resistance genes is an effective counter to diseases of crop plants, the constant evolution of resistant pathogens necessitates ongoing, persistent efforts to combat disease. Recent research suggests that mRNA polyadenylation may play a central role in the responses of plants to numerous stresses. A detailed understanding of this role should be useful in devising new tools and strategies by which crop plants might better respond to multiple stresses over a growing season.This research will bring to bear many tools and approaches. Physiological studies of wild-type and mutant plants will be used to connect parts of the molecular machinery of polyadenylation with stress responses. These connections will provide targets that scientists and producers might manipulate to improve crop productivity. Biochemical experiments will provide information into the workings of some of the main players in the process, and will lead to the identification of new strategies for crop improvement through alteration of polyadenylation. Large-scale DNA and RNA sequencing will provide a broad overview of the interplay between polyadenylation and stress.When completed, this research will provide a deeper understanding of the molecular mechanisms that govern mRNA polyadenylation. More importantly, it will provide scientists and producers with new strategies that may be used to enhance the ability of crop plants to withstand disease and environmental stresses (such as salt, heat, and drought stress). These strategies will be useful additions to the toolkit that farmers may use to improve crop productivity.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2499	1040	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;

Keywords

polyadenylation

stress tolerance

disease resistance

Goals / Objectives
Gene expression can be regulated at any of many steps, and is usually regulated at more than one level. The step of gene expression at which a pre-mRNA is processed and polyadenylated is an important one in terms of regulation. Regulation at this point involves a process in which one of a number of potential poly(A) sites is chosen in favor of others (termed alternative polyadenylation, or APA) and can impact the ultimate expression of a gene in a number of ways.APA is a wide-ranging phenomenon in plants. However, many questions remain to be answered regarding scope, mechanism, and outcome. The Hunt laboratory has proposed that a key aspect of APA in plants is the re-directing of transcriptional output into non-productive pathways in response to stresses (and perhaps developmental cues). This role would be part of a larger collective of responses that together redirect gene expression away from "constitutive" genes and enzymes, and towards genes and proteins that act to ameliorate stresses. Several key studies (some completed, some in the process of publication) have confirmed key aspects of this model.In the past few years, research in the Hunt laboratory has focused on characterizations of APA in Arabidopsis and other plants, with the broad goal of defining the scope of the phenomenon. In the coming years, emphasis will be placed more on mechanism, of 3' processing generally and of poly(A) site choice. Accordingly, the research objectives for the upcoming 5-year period focus are:Assessing the effects of a novel chemical inhibitor of polyadenylation on plants.Functional characterization of CPSF30 in planta and in vitro.Defining the connections between stress-induced alternative polyadenylation and the physiological responses of plants to abiotic stress.

Project Methods
To confirm that AN3661 does on fact inhibit mRNA polyadenylation in Arabidopsis, two genome-wide studies will be conducted in parallel. One of these will entail a study of poly(A) site choice in AN3661-treated plants, using the standard approaches in routine use in the lab. In parallel, all RNA 3' ends (polyadenylated as well as unadenylated) will be profiled using modifications of the standard poly(A) tag library protocol. Thus, a DNA adapter will be ligated to the 3' ends of total Arabidopsis RNA using a truncated T4 RNA ligase and a pre-adenylated, 3'-blocked DNA oligonucleotide. This adapter will serve as a priming site for reverse transcriptase, and the "adapted" RNA will be processed for 3' end tag libraries as is routinely done for poly(A) tags. So-called "GroSeq" experiments will examine the effects of AN3661 (and inhibition of mRNA polyadenylation) on the dynamics of RNA polymerase II. GroSeq entails the characterization of nucleus-localized nascent RNAs, and serves as a means by which transcriptionally-engaged RNA polymerase II may be studied. Using these approaches, the effects of AN3661 on RNA polymerase II dynamics (especially transcription initiation and processivity) will be studied.To isolated AN3661-resistant mutants, plants that can germinate and grow on inhibitory concentrations of AN3661 will be recovered and further analyzed. For this, a panel of EMS-mutagenized pools provided by Dr. Pradeep Kachroo will be screened. Selfed-progeny will be screened to confirm the resistant phenotype; this will both confirm the resistant phenotype and distinguish between mutants that are homozygous or heterozygous. Dominance (or recessiveness) will be tested by crossing confirmed mutants with the wild-type parent. The genomes of individual confirmed mutants will be sequenced, and the question of whether the CPSF73 gene in mutants carries changes that may explain the phenotype will be addressed.To study CPSF30 activities, a tethering assay will be developed using in vitro approaches and used for in vivo studies. Purified MS2 coat protein to which a FLAG tag has been added will be mixed with the purified CPSF30 RNA-binding domain to which a myc epitope has been added. (Protein expression and purification will utilize E. coli expression systems as well as in vitro, coupled transcription and translation systems that are inherently RNAse-free.) Interactions between these two proteins will be studied by co-precipitation using affinity tools specific for the FLAG tag or myc epitope. Assays will be conducted in the absence or presence of the MS2CP-CaMV NUE RNA, as well as RNAs lacking the MS2 CP binding site. As controls, mutant forms of the MS2 CP and CPSF30 RNA binding domain that do not bind RNA will be used.Should the MS2CP tethering approach succeed in the in vitro studies described in the preceding subsection, it will be adapted for the development of an in vivo reporting system that may be used to monitor RNA binding by CPSF30 in vivo. A sequence-specific DNA binding domain (derived from the GAL4 transcriptional activator) will be fused to the MS2 coat protein (and the FLAG tag described above will be retained), and a universal transcriptional activation domain (again, from the yeast GAL4 transcriptional activator) will be fused to various myc-CPSF30 isoforms. These will be introduced into yeast and plants (both Arabidopsis and N. benthamiana) along with a reporter construct that consists of a luciferase coding region driven by a GAL4-responsive promoter, and also transgenes that encode RNAs analogous to those described in the preceding subsection (e.g., RNAs with normal or mutant MS2CP binding sites, and with different possible poly(A) signals). In this experimental design, RNA binding by CPSF30 will be manifest as expression of the luciferase reporter genes, that can be monitored using a standard and sensitive assay. Numerous controls (immunoblots to monitor expression of the tagged MS2CP and CPSF30 isoforms, RT/PCR to confirm expression of the tethering RNA species) will be conducted to confirm the expression of the different components of the system.To study the relationships between polyadenylation and stress responses, he fip1-2 mutant, along with mutants deficient in CPSF30, CstF64, CstF77, and CPSF100 will be studied. The experimental approaches will be similar for all mutants: they will be subjected to a battery of biotic and abiotic stresses, their responses to stresses will be determined, and global poly(A) site usage will be characterized. To study biotic stress, four different treatments will be performed. One will involve treatment of plants with the N-terminal fragment of flagellin from Pseudomonas syringae pv. tabaci (flg22). Additionally, wild-type Arabidopsis (Columbia) plants will be inoculated with Pseudomonas syringae pv tomato DC3000:AvrRpm1; this pathogen induces a hypersensitive response in the wild-type Columbia background. Finally, plants will be infected with necrotrophic and biotrophic pathogens (Alternaria brassicola and Pseudomonas syringae pv tomato DC3000, respectively). In all cases, suitable controls (mock-inoculations or treatments) will be performed. Treated or control plants will be harvested for RNA isolation after the infection or effects of the respective treatment are established. To study abiotic stresses, five different treatments will be conducted. Specifically, Arabidopsis seedlings will be subjected to drought (no watering until wilting) or heat (32°C for 1 hour), cold [exposure for 6 hours to 0°C], salt (periodic watering with NaCl-containing solutions), and oxidative stresses [treatment with methyl viologen]. These selected treatments represent a representative range of stresses, and the results should provide information about a wide range of abiotic stress responses. The experimental design will be such that generic controls (e.g., treatment with water, or incubation at normal growing temperatures) may be used for different treatments, thus reducing the sequencing costs.

Progress 10/10/19 to 09/30/20

Outputs
Target Audience:The target audience for this project is the community of biologists whose interests intersect with the field of RNA processing and metabolism.Given the nature of some of this research (such as that involving use of a compound that has effects on parasites that affect humans and livestock), this community will extend beyond the plant sciences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for one undergraduate student. The training focused on the refinement of methods to express recombinant proteins in E. coli. In this context, and the adoption of this technology for the study of the CPSF30-CAM1 interaction. How have the results been disseminated to communities of interest?The studies describing the genome-wide effects of abiotic stresses on poly(A) site usage in Sorghum was published in early 2020. This report is reference 6 in the section entitled"What was accomplihsed under these goals?". What do you plan to do during the next reporting period to accomplish the goals?Referring to the Major Goals in the order discussed under the entry "What was accomplished under these goals?": 1.The CPSF73 gene in as many as ten independent AN3661-resistant mutants will be sequenced, to test the hypothesis that resistance is due to alterations in the CPSF73 protein. 2.Having shown that an authentic plant calmodulin isoform in fact can bind CPSF30, the components of the in vitro tethering assay will be assembled. Subsequently, the respective proteins will be isolated from suitably-programmed E. coli and assayed for RNA binding. 3. Additional experiments focusing on the stress responses of the oxt6 and fip1-2 mutants will be conducted. In addition, genes with poly(A) sites whose usage depends on CPSF30 will be identified from previously-published sequence databases, and candidates that may play specific roles in responses to pathogens and abiotic stresses selected for follow-up studies.

Impacts
What was accomplished under these goals? The following describe accomplishments under the three goals stated under "Major Goals": 1. A mutagenized population of Arabidopsis seed was screened for individuals that are resistant to AN3661.Several individuals were identified, with approximately 10% of individual mutagenized pools yielding at least one resistant plant. Resistant individuals were grown, allowed to self-fertilize, and progeny seed collected. 2.The interaction between CPSF30 and calmodulin was confirmed, using an Arabidopsis calmodulin isoform (CAM1). For this, a maltose binding protein-CPSF30 fusion protein was expressed in E. coli as previously described in experiments conducted in the PI's laboratory[1-4]. In parallel, the Arabidopsis CAM1 isoform was expressed as a fusion with the so-called V5 tag[5]. Interactions between the two proteins were assayed by measuring co-purification of the V5-CAM1 protein with MBP-CPSF30 using amylose affinity media. As expected, there was an interaction that was not seen with MBP alone, an interaction that was seen in Ca-containing buffer but not in the presence of EGTA. This is an important result, as the CPSF30-CAM interaction had been seen previously with mammalian CAM isoforms, but not with plant CAMs. In the course of this research, a novel method was developed for the expression of recombinant proteins in E. coli. Specifically, an integrated RT/PCR strategy was devised to express proteins using the so-called PURExpress system (New England Biolabs), and the resulting templates subcloned into the pGEM T-Easy system (Promega). These templates are self-contained transcription units that require T7 RNA polymerase; when introduced into cells such as Rosetta cells, high levels of expression can be achieved. This system was used for the CAM1-V5 expression. 3.A large study describing the global effects of abiotic stresses on poly(A) site choice in sorghum was published in early 2020[6]. The key findings reported were that different abiotic stresses (drought, salt, or heat) all trigger a global remodeling of poly(A)site choice in sorghum, the result of which is a significant re-directing of transcriptional output from productive to non-productive outcomes. Based on these findings, we propose that alternative polyadenylation constitutes an important means by which plants re-model gene expression in response to stresses. It was found that the Arabidopsis CPSF30 mutant (oxt6) was impaired in systemic acquired resistance; this finding came from a new collaboration with Dr. Pradeep Kachroo (Dept. of Plant Pathology, University of Kentucky). This finding corroborates an earlier report that noted diminished immune responses in the oxt6 mutant[7], and provides mechanistic insight into the earlier report. 1.Addepalli, B. and A.G. Hunt,A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor.Nucleic Acids Res, 2007.35(13): p. 4453-63. 2.Addepalli, B. and A.G. Hunt,Redox and heavy metal effects on the biochemical activities of an Arabidopsis polyadenylation factor subunit.Arch Biochem Biophys, 2008.473(1): p. 88-95. 3.Addepalli, B., P.A. Limbach, and A.G. Hunt,A disulfide linkage in a CCCH zinc finger motif of an Arabidopsis CPSF30 ortholog.FEBS Lett, 2010.584(21): p. 4408-12. 4.Delaney, K., et al.,Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit.Plant Physiol, 2006.140: p. 1507-1521. 5.McLean, P.J., H. Kawamata, and B.T. Hyman,Alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons.Neuroscience, 2001.104(3): p. 901-12. 6.Chakrabarti, M., et al.,Wide-ranging transcriptome remodelling mediated by alternative polyadenylation in response to abiotic stresses in Sorghum.Plant J, 2020.102(5): p. 916-930. 7.Bruggeman, Q., et al.,The Polyadenylation Factor Subunit CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30: A Key Factor of Programmed Cell Death and a Regulator of Immunity in Arabidopsis.Plant Physiol, 2014.165(2): p. 732-746.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Chakrabarti, M., de Lorenzo, L., Abdel-Ghany, S. E., Reddy, A. S. N., and Hunt, A.G. (2020) Wide-ranging transcriptome remodelling mediated by alternative polyadenylation in response to abiotic stresses in Sorghum. Plant J. 2020;102(5):916-930.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Conesa, C. M., Saez, A., Navarro-Neila, S., de Lorenzo, L., Hunt, A. G., Sepulveda, E. B., Baigorri, R., Garcia-Mina, J. M., Zamarre�o, A. M., Sacristan, S., and del Pozo, J. C. (2020) Alternative polyadenylation and salicylic acid modulate root responses to low nitrogen. Plants, 9(2), 251.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Azzouz-Olden, F., Hunt, A.G. & Dinkins, R. (2020) Transcriptome analysis of drought-tolerant sorghum genotype SC56 in response to water stress reveals an oxidative stress defense strategy. Mol Biol Rep 47, 32913303.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hunt, A. G. (2019) mRNA 3 End Formation in Plants: Novel Connections to Growth, Development and Environmental Responses. WIRES RNA, 2019;e1575.
• Type: Book Chapters Status: Published Year Published: 2020 Citation: Wu, X., Hunt, A. G., and Li, Q. Q. (2020) Genome-wide determination of poly(A) sites in Medicago truncatula: evolutionary conservation of alternative poly(A) site choice. In The Model Legume Medicago truncatula. Frans J. de Bruijn, ed. Wiley/Blackwell. Chapter 12.6.3.