103 results on '"Simpson GG"'
Search Results
2. U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs.
- Author
-
Shen A, Hencel K, Parker MT, Scott R, Skukan R, Adesina AS, Metheringham CL, Miska EA, Nam Y, Haerty W, Simpson GG, and Akay A
- Subjects
- Animals, Humans, Adenosine analogs & derivatives, Adenosine metabolism, Adenosine genetics, Alternative Splicing, Spliceosomes metabolism, Spliceosomes genetics, RNA, Small Nuclear genetics, RNA, Small Nuclear metabolism, Caenorhabditis elegans genetics, Caenorhabditis elegans metabolism, RNA Precursors metabolism, RNA Precursors genetics, Methyltransferases metabolism, Methyltransferases genetics, Caenorhabditis elegans Proteins genetics, Caenorhabditis elegans Proteins metabolism, RNA Splice Sites, RNA Splicing
- Abstract
pre-mRNA splicing is a critical feature of eukaryotic gene expression. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that the conserved U6 snRNA m6A methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of METT-10 in C. elegans and METTL16 in humans primarily leads to alternative splicing at 5' splice sites with an adenosine at +4 position. In addition, METT-10 is required for splicing of weak 3' cis- and trans-splice sites. We identified a significant overlap between METT-10 and the conserved splicing factor SNRNP27K in regulating 5' splice sites with +4A. Finally, we show that editing endogenous 5' splice site +4A positions to +4U restores splicing to wild-type positions in a mett-10 mutant background, supporting a direct role for U6 snRNA m6A modification in 5' splice site recognition. We conclude that the U6 snRNA m6A modification is important for accurate and efficient pre-mRNA splicing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2024
- Full Text
- View/download PDF
3. Inter-species association mapping links splice site evolution to METTL16 and SNRNP27K.
- Author
-
Parker MT, Fica SM, Barton GJ, and Simpson GG
- Subjects
- Adenosine metabolism, Base Sequence, Introns genetics, RNA Precursors metabolism, RNA Splicing, Humans, RNA Splice Sites, RNA, Small Nuclear genetics
- Abstract
Eukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. Patterns of splicing complexity differ between species, but it is unclear how these differences arise. We used inter-species association mapping with Saccharomycotina species to correlate splicing signal phenotypes with the presence or absence of splicing factors. Here, we show that variation in 5' splice site sequence preferences correlate with the presence of the U6 snRNA N6-methyladenosine methyltransferase METTL16 and the splicing factor SNRNP27K. The greatest variation in 5' splice site sequence occurred at the +4 position and involved a preference switch between adenosine and uridine. Loss of METTL16 and SNRNP27K orthologs, or a single SNRNP27K methionine residue, was associated with a preference for +4 U. These findings are consistent with splicing analyses of mutants defective in either METTL16 or SNRNP27K orthologs and models derived from spliceosome structures, demonstrating that inter-species association mapping is a powerful orthogonal approach to molecular studies. We identified variation between species in the occurrence of two major classes of 5' splice sites, defined by distinct interaction potentials with U5 and U6 snRNAs, that correlates with intron number. We conclude that variation in concerted processes of 5' splice site selection by U6 snRNA is associated with evolutionary changes in splicing signal phenotypes., Competing Interests: MP, SF, GB, GS No competing interests declared, (© 2023, Parker et al.)
- Published
- 2023
- Full Text
- View/download PDF
4. U6 snRNA m6A modification is required for accurate and efficient cis- and trans-splicing of C. elegans mRNAs.
- Author
-
Shen A, Hencel K, Parker MT, Scott R, Skukan R, Adesina AS, Metheringham CL, Miska EA, Nam Y, Haerty W, Simpson GG, and Akay A
- Abstract
pre-mRNA splicing is a critical feature of eukaryotic gene expression. Many eukaryotes use cis-splicing to remove intronic sequences from pre-mRNAs. In addition to cis-splicing, many organisms use trans-splicing to replace the 5' ends of mRNAs with a non-coding spliced-leader RNA. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that m6A modification of U6 snRNA A43 by the RNA methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of U6 snRNA m6A modification primarily leads to alternative splicing at 5' splice sites. Furthermore, weaker 5' splice site recognition by the unmodified U6 snRNA A43 affects splicing at 3' splice sites. U6 snRNA m6A43 and the splicing factor SNRNP27K function to recognise an overlapping set of 5' splice sites with an adenosine at +4 position. Finally, we show that U6 snRNA m6A43 is required for efficient SL trans-splicing at weak 3' trans-splice sites. We conclude that the U6 snRNA m6A modification is important for accurate and efficient cis- and trans-splicing in C. elegans .
- Published
- 2023
- Full Text
- View/download PDF
5. Strategies for Conditional Regulation of Proteins.
- Author
-
Nadendla K, Simpson GG, Becher J, Journeaux T, Cabeza-Cabrerizo M, and Bernardes GJL
- Abstract
Design of the next-generation of therapeutics, biosensors, and molecular tools for basic research requires that we bring protein activity under control. Each protein has unique properties, and therefore, it is critical to tailor the current techniques to develop new regulatory methods and regulate new proteins of interest (POIs). This perspective gives an overview of the widely used stimuli and synthetic and natural methods for conditional regulation of proteins., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)
- Published
- 2023
- Full Text
- View/download PDF
6. m 6 A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site.
- Author
-
Parker MT, Soanes BK, Kusakina J, Larrieu A, Knop K, Joy N, Breidenbach F, Sherwood AV, Barton GJ, Fica SM, Davies BH, and Simpson GG
- Subjects
- Humans, RNA Splice Sites, RNA Splicing, RNA, Small Nuclear genetics, Spliceosomes metabolism, RNA Precursors metabolism, Arabidopsis genetics, Arabidopsis metabolism
- Abstract
Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the AC A GA box is modified by N
6 methylation, but the role of this m6 A modification is poorly understood. Here, we show that m6 A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m6 A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5' splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6 A influences 3' splice site usage. We generalise these findings to reveal two major classes of 5' splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA AC A GA box. We conclude that U6 snRNA m6 A modification contributes to the selection of degenerate 5' splice sites crucial to alternative splicing., Competing Interests: MP, BS, JK, AL, KK, NJ, FB, AS, GB, SF, BD, GS No competing interests declared, (© 2022, Parker et al.)- Published
- 2022
- Full Text
- View/download PDF
7. Chromosome evolution and the genetic basis of agronomically important traits in greater yam.
- Author
-
Bredeson JV, Lyons JB, Oniyinde IO, Okereke NR, Kolade O, Nnabue I, Nwadili CO, Hřibová E, Parker M, Nwogha J, Shu S, Carlson J, Kariba R, Muthemba S, Knop K, Barton GJ, Sherwood AV, Lopez-Montes A, Asiedu R, Jamnadass R, Muchugi A, Goodstein D, Egesi CN, Featherston J, Asfaw A, Simpson GG, Doležel J, Hendre PS, Van Deynze A, Kumar PL, Obidiegwu JE, Bhattacharjee R, and Rokhsar DS
- Subjects
- Chromosomes, Humans, Plant Breeding, Plant Tubers, Quantitative Trait Loci genetics, Dioscorea genetics
- Abstract
The nutrient-rich tubers of the greater yam, Dioscorea alata L., provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an orphan crop. Here, we address this resource gap by presenting a highly contiguous chromosome-scale genome assembly of D. alata combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient allotetraploidization in the Dioscorea lineage, followed by extensive genome-wide reorganization. Using the genomic tools, we find quantitative trait loci for resistance to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
- Published
- 2022
- Full Text
- View/download PDF
8. Two zinc finger proteins with functions in m 6 A writing interact with HAKAI.
- Author
-
Zhang M, Bodi Z, Mackinnon K, Zhong S, Archer N, Mongan NP, Simpson GG, and Fray RG
- Subjects
- Animals, Methylation, RNA, Messenger genetics, RNA, Messenger metabolism, Writing, Zinc Fingers, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism
- Abstract
The methyltransferase complex (m
6 A writer), which catalyzes the deposition of N6 -methyladenosine (m6 A) in mRNAs, is highly conserved across most eukaryotic organisms, but its components and interactions between them are still far from fully understood. Here, using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, are discovered as components of the Arabidopsis m6 A writer complex. HAKAI is required for the interaction between HIZ1 and MTA (mRNA adenosine methylase A). Whilst HIZ1 knockout plants have normal levels of m6 A, plants in which it is overexpressed show reduced methylation and decreased lateral root formation. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m6 A abundance and show severe developmental defects. Our findings suggest that HIZ2 is likely the plant equivalent of ZC3H13 (Flacc) of the metazoan m6 A-METTL Associated Complex., (© 2022. The Author(s).)- Published
- 2022
- Full Text
- View/download PDF
9. Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA.
- Author
-
Parker MT, Knop K, Zacharaki V, Sherwood AV, Tomé D, Yu X, Martin PG, Beynon J, Michaels SD, Barton GJ, and Simpson GG
- Subjects
- Arabidopsis metabolism, Arabidopsis Proteins genetics, Gene Expression Regulation, Plant, Genes, Plant genetics, Genes, Plant physiology, RNA, Messenger metabolism, Arabidopsis genetics, Arabidopsis Proteins metabolism, NLR Proteins metabolism, RNA-Binding Proteins metabolism, Transcription Termination, Genetic
- Abstract
Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers of nucleotide-binding, leucine-rich repeat (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here, we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants., Competing Interests: MP, KK, VZ, AS, DT, XY, PM, JB, SM, GB, GS No competing interests declared, (© 2021, Parker et al.)
- Published
- 2021
- Full Text
- View/download PDF
10. 2passtools: two-pass alignment using machine-learning-filtered splice junctions increases the accuracy of intron detection in long-read RNA sequencing.
- Author
-
Parker MT, Knop K, Barton GJ, and Simpson GG
- Subjects
- Introns, Molecular Sequence Annotation, RNA Splicing, Reproducibility of Results, Algorithms, Computational Biology methods, Machine Learning, RNA Splice Sites, RNA-Seq methods, Sequence Alignment methods, Software
- Abstract
Transcription of eukaryotic genomes involves complex alternative processing of RNAs. Sequencing of full-length RNAs using long reads reveals the true complexity of processing. However, the relatively high error rates of long-read sequencing technologies can reduce the accuracy of intron identification. Here we apply alignment metrics and machine-learning-derived sequence information to filter spurious splice junctions from long-read alignments and use the remaining junctions to guide realignment in a two-pass approach. This method, available in the software package 2passtools ( https://github.com/bartongroup/2passtools ), improves the accuracy of spliced alignment and transcriptome assembly for species both with and without existing high-quality annotations.
- Published
- 2021
- Full Text
- View/download PDF
11. Multifunctional opioid receptor agonism and antagonism by a novel macrocyclic tetrapeptide prevents reinstatement of morphine-seeking behaviour.
- Author
-
Brice-Tutt AC, Wilson LL, Eans SO, Stacy HM, Simons CA, Simpson GG, Coleman JS, Ferracane MJ, Aldrich JV, and McLaughlin JP
- Subjects
- Analgesics, Opioid pharmacology, Animals, Mice, Mice, Inbred C57BL, Narcotic Antagonists, Receptors, Opioid, Morphine pharmacology, Pharmaceutical Preparations, Receptors, Opioid, mu agonists, Receptors, Opioid, mu antagonists & inhibitors
- Abstract
Background and Purpose: The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) is a multifunctional μ-opioid receptor and κ-opioid receptor agonist and κ-opioid receptor antagonist that produces antinociception and prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference (CPP). We hypothesized that an analogue of CJ-15,208, cyclo[Pro-Sar-Phe-d-Phe], would demonstrate multifunctional μ-opioid receptor and κ-opioid receptor ligand activity, producing potent antinociception with fewer liabilities than selective μ-opioid receptor agonists, while preventing both drug- and stress-induced reinstatement of morphine-induced CPP., Experimental Approach: The opioid receptor agonist and antagonist activity of cyclo[Pro-Sar-Phe-d-Phe] was characterized after i.c.v. and i.p. administration to C57BL/6J or transgenic opioid receptor "knockout" mice using the 55°C warm-water tail-withdrawal assay. Liabilities of locomotor coordination, respiration and spontaneous ambulation, and direct rewarding or aversive properties were assessed. Finally, the ability of cyclo[Pro-Sar-Phe-d-Phe] to block morphine- and stress-induced reinstatement of extinguished CPP was determined., Key Results: cyclo[Pro-Sar-Phe-d-Phe] demonstrated dose-dependent, short-lasting antinociception, with an ED
50 (and 95% confidence interval) of 0.15 (0.05-0.21) nmol i.c.v. and 1.91 (0.40-3.54) mg·kg-1 i.p., mediated by μ- and κ-opioid receptors. The macrocyclic tetrapeptide also demonstrated potent dose-dependent κ-opioid receptor antagonist-like activity at 2.5, but not at 4.5, h after administration. cyclo[Pro-Sar-Phe-d-Phe] displayed reduced liabiities compared with morphine, attributed to its additional activity at κ-receptors. Pretreatment with cyclo[Pro-Sar-Phe-d-Phe] prevented stress- and drug-induced reinstatement of extinguished morphine-place preference responses in a time-dependent manner., Conclusions and Implications: These data suggest that cyclo[Pro-Sar-Phe-d-Phe] is a promising lead compound for both the treatment of pain with reduced sideeffects and preventing both drug- and stress-induced relapse in morphine-abstinent subjects., (© 2020 The British Pharmacological Society.)- Published
- 2020
- Full Text
- View/download PDF
12. Design, Synthesis, and Characterization of the Macrocyclic Tetrapeptide cyclo [Pro-Sar-Phe-d-Phe]: A Mixed Opioid Receptor Agonist-Antagonist Following Oral Administration.
- Author
-
Ferracane MJ, Brice-Tutt AC, Coleman JS, Simpson GG, Wilson LL, Eans SO, Stacy HM, Murray TF, McLaughlin JP, and Aldrich JV
- Subjects
- Administration, Oral, Animals, Mice, Mice, Inbred C57BL, Oligopeptides, Peptides, Cyclic, Receptors, Opioid, kappa, Receptors, Opioid, mu, Analgesics, Opioid, Narcotic Antagonists
- Abstract
Substance abuse remains a serious public health crisis, affecting millions of people worldwide. Macrocyclic tetrapeptides like CJ-15,208 and [d-Trp]CJ-15,208 demonstrate opioid activity shown to attenuate the rewarding effects of cocaine in conditioned place preference assays in mice, making them promising lead compounds for treating substance abuse. In the present study, we report the rational design, synthesis, conformational analysis, and continued pharmacological evaluation of the novel macrocyclic tetrapeptide cyclo [Pro-Sar-Phe-d-Phe] to further explore this unique molecular scaffold. This peptide was rationally designed based on X-ray and NMR structures of related macrocyclic tetrapeptides. Following synthesis, its solution-phase conformations were determined by NMR and molecular modeling. The peptide adopted multiple conformations in polar solvents, but a single conformation in chloroform that is stabilized by intramolecular hydrogen bonding. The peptide is orally bioavailable, producing antinociception and antagonism of kappa opioid receptor (KOR) stimulation following oral administration in a mouse 55 °C warm-water tail-withdrawal assay. Notably, cyclo [Pro-Sar-Phe-d-Phe] blocked both stress- and drug-induced reinstatement of cocaine and morphine conditioned place preference in mice following oral administration, and displayed a decreased side-effect profile compared to morphine. Thus, cyclo [Pro-Sar-Phe-d-Phe] is a promising lead compound for the treatment of substance abuse.
- Published
- 2020
- Full Text
- View/download PDF
13. Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m 6 A modification.
- Author
-
Parker MT, Knop K, Sherwood AV, Schurch NJ, Mackinnon K, Gould PD, Hall AJ, Barton GJ, and Simpson GG
- Subjects
- Adenosine metabolism, Arabidopsis metabolism, Gene Expression Profiling, Methylation, Nanopores, Poly A genetics, Poly A metabolism, RNA Caps, RNA Splicing, RNA, Messenger chemistry, RNA, Messenger metabolism, RNA, Plant chemistry, RNA, Plant metabolism, RNA, Untranslated chemistry, RNA, Untranslated genetics, Adenosine analogs & derivatives, Arabidopsis genetics, RNA Processing, Post-Transcriptional, RNA, Messenger genetics, RNA, Plant genetics, Sequence Analysis, RNA
- Abstract
Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m
6 A). Here we show that m6 A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6 A from 3' untranslated regions is associated with decreased relative transcript abundance and defective RNA 3' end formation. A functional consequence of disrupted m6 A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode., Competing Interests: MP, KK, AS, NS, KM, PG, AH, GB, GS No competing interests declared, (© 2020, Parker et al.)- Published
- 2020
- Full Text
- View/download PDF
14. How well do RNA-Seq differential gene expression tools perform in a complex eukaryote? A case study in Arabidopsis thaliana.
- Author
-
Froussios K, Schurch NJ, Mackinnon K, Gierliński M, Duc C, Simpson GG, and Barton GJ
- Subjects
- Binomial Distribution, RNA-Seq, Sequence Analysis, RNA, Software, Arabidopsis
- Abstract
Motivation: RNA-seq experiments are usually carried out in three or fewer replicates. In order to work well with so few samples, differential gene expression (DGE) tools typically assume the form of the underlying gene expression distribution. In this paper, the statistical properties of gene expression from RNA-seq are investigated in the complex eukaryote, Arabidopsis thaliana, extending and generalizing the results of previous work in the simple eukaryote Saccharomyces cerevisiae., Results: We show that, consistent with the results in S.cerevisiae, more gene expression measurements in A.thaliana are consistent with being drawn from an underlying negative binomial distribution than either a log-normal distribution or a normal distribution, and that the size and complexity of the A.thaliana transcriptome does not influence the false positive rate performance of nine widely used DGE tools tested here. We therefore recommend the use of DGE tools that are based on the negative binomial distribution., Availability and Implementation: The raw data for the 17 WT Arabidopsis thaliana datasets is available from the European Nucleotide Archive (E-MTAB-5446). The processed and aligned data can be visualized in context using IGB (Freese et al., 2016), or downloaded directly, using our publicly available IGB quickload server at https://compbio.lifesci.dundee.ac.uk/arabidopsisQuickload/public_quickload/ under 'RNAseq>Froussios2019'. All scripts and commands are available from github at https://github.com/bartongroup/KF_arabidopsis-GRNA., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press.)
- Published
- 2019
- Full Text
- View/download PDF
15. Incorporation of the HEART Score Into a Low-risk Chest Pain Pathway to Safely Decrease Admissions.
- Author
-
Allen BR, Simpson GG, Zeinali I, Freitas JT, Chapa JJ, Rawson LJ, Richter JA, Payton TF, and Tyndall JA
- Subjects
- Chest Pain therapy, Electrocardiography, Female, Florida, Follow-Up Studies, Humans, Male, Middle Aged, Retrospective Studies, Risk Factors, Time Factors, Chest Pain diagnosis, Decision Making, Emergency Service, Hospital statistics & numerical data, Patient Admission trends, Risk Assessment methods, Triage standards
- Abstract
Chest pain can be a challenging complaint to manage in the emergency department. A missed diagnosis can result in significant morbidity or mortality, whereas avoidable testing and hospitalizations can lead to increased health care costs, contribute to hospital crowding, and increase risks to patients. The HEART score is a validated decision aid to identify patients at low risk for acute coronary syndrome who can be safely discharged without admission or objective cardiac testing. In the largest and one of the longest studies to date (N = 31,060; 30 months), we included the HEART score into a larger, newly developed low-risk chest pain decision pathway, using a retrospective observational pre/post study design with the objective of safely lowering admissions. The modified HEART score calculation tool was incorporated in our electronic medical record. A significant increase in discharges of low-risk chest pain patients (relative increase of 21%; p < 0.0001) in the postimplementation period was observed with no significant difference in the rates of major adverse cardiac events between the pre and post periods. There was a decrease in the amount of return admissions for 30 days (4.65% fewer; p = 0.009) and 60 days (3.78% fewer; p = 0.020). No significant difference in length of stay was observed for patients who were ultimately discharged. A 64% decrease in monthly coronary computed tomography angiograms was observed in the post period (p < 0.0001). These findings support the growing consensus in the literature that the adoption of the HEART pathway or similar protocols in emergency departments, including at large and high-volume medical institutions, can substantially benefit patient care and reduce associated health care costs.
- Published
- 2018
- Full Text
- View/download PDF
16. A Patient-Centered Emergency Department Management Strategy for Sickle-Cell Disease Super-Utilizers.
- Author
-
Simpson GG, Hahn HR, Powel AA, Leverence RR, Morris LA, Thompson LG, Zumberg MS, Borde DJ, Tyndall JA, Shuster JJ, Yealy DM, and Allen BR
- Subjects
- Adult, Ambulatory Care economics, Ambulatory Care organization & administration, Anemia, Sickle Cell economics, Antisickling Agents economics, Blood Transfusion, Cost-Benefit Analysis, Emergency Service, Hospital economics, Feasibility Studies, Female, Florida, Health Services Accessibility, Health Services Misuse economics, Humans, Interdisciplinary Communication, Male, Patient Acceptance of Health Care, Pilot Projects, Ambulatory Care statistics & numerical data, Anemia, Sickle Cell therapy, Antisickling Agents therapeutic use, Emergency Service, Hospital organization & administration, Emergency Service, Hospital statistics & numerical data, Health Services Misuse prevention & control, Patient-Centered Care economics, Patient-Centered Care organization & administration
- Abstract
Introduction: A subpopulation of sickle-cell disease patients, termed super-utilizers, presents frequently to emergency departments (EDs) for vaso-occlusive events and may consume disproportionate resources without broader health benefit. To address the healthcare needs of this vulnerable patient population, we piloted a multidisciplinary intervention seeking to create and use individualized patient care plans that alter utilization through coordinated care. Our goals were to assess feasibility primarily, and to assess resource use secondarily., Methods: We evaluated the effects of a single-site interventional study targeted at a population of adult sickle-cell disease super-utilizers using a pre- and post-implementation design. The pre-intervention period was 06/01/13 to 12/31/13 (seven months) and the post-intervention period was 01/01/14 to 02/28/15 (14 months). Our approach included patient-specific best practice advisories (BPA); an ED management protocol; and formation of a "medical home" for these patients., Results: For 10 subjects targeted initially we developed and implemented coordinated care plans; after deployment, we observed a tendency toward reduction in ED and inpatient utilization across all measured indices. Between the annualized pre- and post-implementation periods we found the following: ED visits decreased by 16.5 visits/pt-yr (95% confidence interval [CI] [-1.32-34.2]); ED length of state (LOS) decreased by 115.3 hours/pt-yr (95% CI [-82.9-313.5]); in-patient admissions decreased by 4.20 admissions/pt-yr (95% CI [-1.73-10.1]); in-patient LOS decreased by 35.8 hours/pt-yr (95% CI [-74.9-146.7]); and visits where the patient left before treatment were reduced by an annualized total of 13.7 visits. We observed no patient mortality in our 10 subjects, and no patient required admission to the intensive care unit 72 hours following discharge., Conclusion: This effort suggests that a targeted approach is both feasible and potentially effective, laying a foundation for broader study., Competing Interests: Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. Research reported in this publication was partly supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001427. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
- Published
- 2017
- Full Text
- View/download PDF
17. Erratum: How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?
- Author
-
Schurch NJ, Schofield P, Gierliński M, Cole C, Sherstnev A, Singh V, Wrobel N, Gharbi K, Simpson GG, Owen-Hughes T, Blaxter M, and Barton GJ
- Published
- 2016
- Full Text
- View/download PDF
18. Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA.
- Author
-
Zhang Y, Rataj K, Simpson GG, and Tong L
- Subjects
- Arabidopsis Proteins physiology, Conserved Sequence, Crystallography, X-Ray, Models, Molecular, Protein Domains, RNA-Binding Proteins physiology, Sequence Alignment, Sequence Analysis, Protein, Arabidopsis Proteins chemistry, RNA-Binding Proteins chemistry
- Abstract
The Arabidopsis protein FPA controls flowering time by regulating the alternative 3'-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3'-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.
- Published
- 2016
- Full Text
- View/download PDF
19. How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?
- Author
-
Schurch NJ, Schofield P, Gierliński M, Cole C, Sherstnev A, Singh V, Wrobel N, Gharbi K, Simpson GG, Owen-Hughes T, Blaxter M, and Barton GJ
- Subjects
- Gene Expression Profiling, RNA, Fungal genetics, Reproducibility of Results, Saccharomyces cerevisiae genetics, Sequence Analysis, RNA methods
- Abstract
RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools., (© 2016 Schurch et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2016
- Full Text
- View/download PDF
20. The Arabidopsis epitranscriptome.
- Author
-
Fray RG and Simpson GG
- Subjects
- Arabidopsis metabolism, Methylation, RNA, Messenger metabolism, Transcriptome, Arabidopsis genetics, Epigenesis, Genetic, Gene Expression Regulation, Plant, RNA, Messenger genetics
- Abstract
The most prevalent internal modification of plant messenger RNAs, N(6)-methyladenosine (m(6)A), was first discovered in the 1970s, then largely forgotten. However, the impact of modifications to eukaryote mRNA, collectively known as the epitranscriptome, has recently attracted renewed attention. mRNA methylation is required for normal Arabidopsis development and the first methylation maps reveal that thousands of Arabidopsis mRNAs are methylated. Arabidopsis is likely to be a model of wide utility in understanding the biological impacts of the epitranscriptome. We review recent progress and look ahead with questions awaiting answers to reveal an entire layer of gene regulation that has until recently been overlooked., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
21. Enter exitrons.
- Author
-
Staiger D and Simpson GG
- Subjects
- Female, Humans, Alternative Splicing, Exons, Introns, Open Reading Frames, Proteomics
- Abstract
Exitrons are exon-like introns located within protein-coding exons. Removal or retention of exitrons through alternative splicing increases proteome complexity and thus adds to phenotypic diversity.
- Published
- 2015
- Full Text
- View/download PDF
22. Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-Seq and ESTs.
- Author
-
Schurch NJ, Cole C, Sherstnev A, Song J, Duc C, Storey KG, McLean WH, Brown SJ, Simpson GG, and Barton GJ
- Subjects
- Animals, Arabidopsis genetics, Cell Cycle Proteins genetics, Chickens genetics, Genetic Loci genetics, Genome, Genome, Human, Genome, Plant, Genomics, Humans, Methyltransferases genetics, Models, Statistical, RNA, Messenger metabolism, Ribosomal Proteins genetics, Skin metabolism, 3' Untranslated Regions, Computational Biology methods, Expressed Sequence Tags, Sequence Analysis, RNA methods
- Abstract
The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3' untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3' polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3' UTR re-annotation (including extension of one 3' UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.
- Published
- 2014
- Full Text
- View/download PDF
23. Message ends: RNA 3' processing and flowering time control.
- Author
-
Rataj K and Simpson GG
- Subjects
- Plant Proteins genetics, Plant Proteins metabolism, RNA, Antisense genetics, RNA, Antisense metabolism, RNA, Messenger metabolism, Time Factors, Flowers genetics, Flowers physiology, RNA 3' End Processing genetics, RNA, Messenger genetics
- Abstract
Plants control the time at which they flower in order to ensure reproductive success. This control is underpinned by precision in gene regulation acting through genetically separable pathways. The genetic dissection of this process in the model plant Arabidopsis thaliana has led to the recurrent identification of plant-specific and highly conserved RNA 3' end processing factors required to control flowering by specifically controlling transcription of mRNA encoding the floral repressor FLOWERING LOCUS C (FLC). Here, we review the features of these RNA-processing and RNA-associated proteins, and the complex architecture of coding and non-coding RNA transcription at the FLC locus. We discuss alternative concepts that might explain how these RNA-processing events regulate FLC transcription and hence control flowering time.
- Published
- 2014
- Full Text
- View/download PDF
24. The RNA-binding protein FPA regulates flg22-triggered defense responses and transcription factor activity by alternative polyadenylation.
- Author
-
Lyons R, Iwase A, Gänsewig T, Sherstnev A, Duc C, Barton GJ, Hanada K, Higuchi-Takeuchi M, Matsui M, Sugimoto K, Kazan K, Simpson GG, and Shirasu K
- Subjects
- Arabidopsis drug effects, Arabidopsis genetics, Arabidopsis growth & development, Immunity, Innate genetics, Peptide Fragments pharmacology, Plant Diseases genetics, Plants, Genetically Modified drug effects, Plants, Genetically Modified genetics, Plants, Genetically Modified growth & development, Pseudomonas syringae genetics, Pseudomonas syringae growth & development, Pseudomonas syringae pathogenicity, RNA, Messenger genetics, RNA, Plant genetics, RNA-Binding Proteins genetics, Reactive Oxygen Species metabolism, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Flagellin pharmacology, Gene Expression Regulation, Plant, Plant Diseases microbiology, Polyadenylation genetics, RNA Processing, Post-Transcriptional, RNA-Binding Proteins metabolism, Repressor Proteins genetics
- Abstract
RNA-binding proteins (RBPs) play an important role in plant host-microbe interactions. In this study, we show that the plant RBP known as FPA, which regulates 3'-end mRNA polyadenylation, negatively regulates basal resistance to bacterial pathogen Pseudomonas syringae in Arabidopsis. A custom microarray analysis reveals that flg22, a peptide derived from bacterial flagellins, induces expression of alternatively polyadenylated isoforms of mRNA encoding the defence-related transcriptional repressor ETHYLENE RESPONSE FACTOR 4 (ERF4), which is regulated by FPA. Flg22 induces expression of a novel isoform of ERF4 that lacks the ERF-associated amphiphilic repression (EAR) motif, while FPA inhibits this induction. The EAR-lacking isoform of ERF4 acts as a transcriptional activator in vivo and suppresses the flg22-dependent reactive oxygen species burst. We propose that FPA controls use of proximal polyadenylation sites of ERF4, which quantitatively limit the defence response output.
- Published
- 2013
- Full Text
- View/download PDF
25. Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.
- Author
-
Duc C, Sherstnev A, Cole C, Barton GJ, and Simpson GG
- Subjects
- Alternative Splicing genetics, Arabidopsis Proteins biosynthesis, Arabidopsis Proteins metabolism, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, DNA Methylation genetics, Exons, Gene Silencing, Heterochromatin genetics, Jumonji Domain-Containing Histone Demethylases genetics, Jumonji Domain-Containing Histone Demethylases metabolism, Mutation, RNA, Messenger genetics, RNA-Binding Proteins biosynthesis, RNA-Binding Proteins metabolism, Ribonuclease III genetics, Ribonuclease III metabolism, Arabidopsis genetics, Arabidopsis Proteins genetics, RNA, Messenger biosynthesis, RNA-Binding Proteins genetics, Transcription Termination, Genetic
- Abstract
Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3' formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3' end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3' end formation can have on gene and genome organisation., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2013
- Full Text
- View/download PDF
26. Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis.
- Author
-
Vanholme R, Cesarino I, Rataj K, Xiao Y, Sundin L, Goeminne G, Kim H, Cross J, Morreel K, Araujo P, Welsh L, Haustraete J, McClellan C, Vanholme B, Ralph J, Simpson GG, Halpin C, and Boerjan W
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins genetics, Carboxylic Ester Hydrolases genetics, Glucose chemistry, Metabolic Networks and Pathways, Mutation, Shikimic Acid chemistry, Substrate Specificity, Arabidopsis enzymology, Arabidopsis Proteins chemistry, Carboxylic Ester Hydrolases chemistry, Lignin biosynthesis
- Abstract
Lignin is a major component of plant secondary cell walls. Here we describe caffeoyl shikimate esterase (CSE) as an enzyme central to the lignin biosynthetic pathway. Arabidopsis thaliana cse mutants deposit less lignin than do wild-type plants, and the remaining lignin is enriched in p-hydroxyphenyl units. Phenolic metabolite profiling identified accumulation of the lignin pathway intermediate caffeoyl shikimate in cse mutants as compared to caffeoyl shikimate levels in the wild type, suggesting caffeoyl shikimate as a substrate for CSE. Accordingly, recombinant CSE hydrolyzed caffeoyl shikimate into caffeate. Associated with the changes in lignin, the conversion of cellulose to glucose in cse mutants increased up to fourfold as compared to that in the wild type upon saccharification without pretreatment. Collectively, these data necessitate the revision of currently accepted models of the lignin biosynthetic pathway.
- Published
- 2013
- Full Text
- View/download PDF
27. Direct sequencing of Arabidopsis thaliana RNA reveals patterns of cleavage and polyadenylation.
- Author
-
Sherstnev A, Duc C, Cole C, Zacharaki V, Hornyik C, Ozsolak F, Milos PM, Barton GJ, and Simpson GG
- Subjects
- Base Sequence, Chromosome Mapping, Gene Expression Regulation, Plant, Genes, Plant, Molecular Sequence Data, Nucleic Acid Conformation, Oligodeoxyribonucleotides metabolism, Polyadenylation, RNA 3' End Processing, RNA, Antisense genetics, RNA, Antisense metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Plant chemistry, RNA, Small Nucleolar genetics, RNA, Small Nucleolar metabolism, RNA, Untranslated chemistry, RNA, Untranslated genetics, RNA, Untranslated metabolism, Sequence Analysis, RNA, Arabidopsis genetics, Arabidopsis metabolism, RNA, Plant genetics, RNA, Plant metabolism
- Abstract
It has recently been shown that RNA 3'-end formation plays a more widespread role in controlling gene expression than previously thought. To examine the impact of regulated 3'-end formation genome-wide, we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and describe the effects of 3'-end formation on genome organization. We reveal extreme heterogeneity in RNA 3' ends, discover previously unrecognized noncoding RNAs and propose widespread reannotation of the genome. We explain the origin of most poly(A)(+) antisense RNAs and identify cis elements that control 3'-end formation in different registers. These findings are essential to understanding what the genome actually encodes, how it is organized and how regulated 3'-end formation affects these processes.
- Published
- 2012
- Full Text
- View/download PDF
28. Noncanonical translation initiation of the Arabidopsis flowering time and alternative polyadenylation regulator FCA.
- Author
-
Simpson GG, Laurie RE, Dijkwel PP, Quesada V, Stockwell PA, Dean C, and Macknight RC
- Subjects
- 5' Untranslated Regions, Arabidopsis Proteins genetics, Base Sequence, Codon, Initiator, Molecular Sequence Data, Open Reading Frames, Plants, Genetically Modified, Point Mutation, Polyadenylation, Promoter Regions, Genetic, RNA Processing, Post-Transcriptional, RNA, Messenger genetics, RNA, Messenger metabolism, RNA-Binding Proteins genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Transcription Initiation Site, Transgenes, Arabidopsis anatomy & histology, Arabidopsis physiology, Arabidopsis Proteins metabolism, Flowers physiology, Gene Expression Regulation, Plant, Protein Biosynthesis, RNA-Binding Proteins metabolism
- Abstract
The RNA binding protein FCA regulates the floral transition and is required for silencing RNAs corresponding to specific noncoding sequences in the Arabidopsis thaliana genome. Through interaction with the canonical RNA 3' processing machinery, FCA affects alternative polyadenylation of many transcripts, including antisense RNAs at the locus encoding the floral repressor FLC. This potential for widespread alteration of gene regulation clearly needs to be tightly regulated, and we have previously shown that FCA expression is autoregulated through poly(A) site choice. Here, we show distinct layers of FCA regulation that involve sequences within the 5' region that regulate noncanonical translation initiation and alter the expression profile. FCA translation in vivo occurs exclusively at a noncanonical CUG codon upstream of the first in-frame AUG. We fully define the upstream flanking sequences essential for its selection, revealing features that distinguish this from other non-AUG start site mechanisms. Bioinformatic analysis identified 10 additional Arabidopsis genes that likely initiate translation at a CUG codon. Our findings reveal further unexpected complexity in the regulation of FCA expression with implications for its roles in regulating flowering time and gene expression and more generally show plant mRNA exceptions to AUG translation initiation.
- Published
- 2010
- Full Text
- View/download PDF
29. Alternative polyadenylation of antisense RNAs and flowering time control.
- Author
-
Hornyik C, Duc C, Rataj K, Terzi LC, and Simpson GG
- Subjects
- Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis metabolism, Flowers growth & development, Flowers metabolism, Gene Expression Regulation, Plant, Models, Biological, RNA Splice Sites physiology, Time Factors, Flowers genetics, Polyadenylation physiology, RNA, Antisense metabolism
- Abstract
Flowering time is controlled by precision in gene regulation mediated by different pathways. Two Arabidopsis thaliana components of the autonomous flowering pathway, FCA and FPA, function as genetically independent trans-acting regulators of alternative cleavage and polyadenylation. FCA and FPA directly associate with chromatin at the locus encoding the floral repressor FLC, but appear to control FLC transcription by mediating alternative polyadenylation of embedded non-coding antisense RNAs. These findings prompt the re-examination of how other factors control FLC expression, as it is formally possible that they function primarily to control alternative processing of antisense RNAs. As co-expressed sense and antisense gene pairs are widespread in eukaryotes, alternative processing of antisense RNAs may represent a significant form of gene regulation.
- Published
- 2010
- Full Text
- View/download PDF
30. The spen family protein FPA controls alternative cleavage and polyadenylation of RNA.
- Author
-
Hornyik C, Terzi LC, and Simpson GG
- Subjects
- Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis metabolism, Base Sequence, Epigenesis, Genetic, Flowers growth & development, Flowers metabolism, Gene Expression Regulation, Plant, Genes, Plant, Mutation, Plants, Genetically Modified, Polyadenylation, RNA Precursors genetics, RNA Precursors metabolism, RNA, Antisense genetics, RNA, Antisense metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Plant genetics, RNA, Plant metabolism, Retroelements, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism
- Abstract
The spen family protein FPA is required for flowering time control and has been implicated in RNA silencing. The mechanism by which FPA carries out these functions is unknown. We report the identification of an activity for FPA in controlling mRNA 3' end formation. We show that FPA functions redundantly with FCA, another RNA binding protein that controls flowering and RNA silencing, to control the expression of alternatively polyadenylated antisense RNAs at the locus encoding the floral repressor FLC. In addition, we show that defective 3' end formation at an upstream RNA polymerase II-dependent gene explains the apparent derepression of the AtSN1 retroelement in fpa mutants. Transcript readthrough accounts for the absence of changes in DNA methylation and siRNA abundance at AtSN1 in fpa mutants, and this may explain other examples of epigenetic transitions not associated with chromatin modification., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
31. Arabidopsis RNA immunoprecipitation.
- Author
-
Terzi LC and Simpson GG
- Subjects
- Arabidopsis metabolism, Arabidopsis Proteins genetics, Gene Expression Regulation, Plant, RNA, Plant metabolism, RNA, Small Nuclear genetics, RNA-Binding Proteins genetics, Arabidopsis genetics, Arabidopsis Proteins metabolism, Immunoprecipitation methods, RNA, Small Nuclear metabolism, RNA-Binding Proteins metabolism
- Abstract
RNA-binding proteins are key regulators of plant gene expression. Consistent with this, the Arabidopsis genome encodes many RNA-binding proteins that are genetically required for normal development and for responding to environmental changes. However, the direct RNA targets and RNA processing events that these RNA-binding proteins control are poorly understood. In order to facilitate the functional characterization of RNA-binding proteins, we have applied the RNA immunoprecipitation assay to Arabidopsis. Working with the U2B''-U2 snRNA interaction as a model experimental system, we show that treatment of intact plants with formaldehyde allows immunocapture of U2 snRNA using antibodies that recognize U2B'' fused to the generic GFP tag. When coupled with recent developments in whole-genome tiling arrays and high-throughput next-generation sequencing, this combination of procedures and technology has the potential to allow systematic functional analysis of plant RNA-binding proteins.
- Published
- 2009
- Full Text
- View/download PDF
32. Regulation of flowering time by RNA processing.
- Author
-
Terzi LC and Simpson GG
- Subjects
- Alternative Splicing, Biological Clocks, Flowers growth & development, MicroRNAs physiology, Plant Proteins physiology, Plants genetics, Plants metabolism, Polyadenylation, RNA 3' End Processing, RNA, Messenger metabolism, RNA-Binding Proteins physiology, Gene Expression Regulation, Plant, Plant Development, RNA, Plant metabolism
- Abstract
Plants control the time at which they flower by integrating environmental cues such as day length and temperature with an endogenous program of development. Flowering time is a quantitative trait and a model for how precision in gene regulation is delivered. In this review, we reveal that flowering time control is particularly rich in RNA processing-based gene regulatory phenomena. We review those factors which function in conserved RNA processing events like alternative 3' end formation, splicing, RNA export and miRNA biogenesis and how they affect flowering time. Likewise, we review the novel plant-specific RNA-binding proteins identified as regulators of flowering time control. In addition, we add to the network of flowering time control pathways, information on alternative processing of flowering time gene pre-mRNAs. Finally, we describe new approaches to dissect the mechanisms which underpin this control.
- Published
- 2008
- Full Text
- View/download PDF
33. An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.
- Author
-
Henderson IR, Liu F, Drea S, Simpson GG, and Dean C
- Subjects
- Arabidopsis growth & development, Arabidopsis Proteins metabolism, Gene Expression Regulation, Plant, Gene Silencing, RNA, Messenger metabolism, RNA-Binding Proteins genetics, Transgenes, Alleles, Arabidopsis genetics, Arabidopsis Proteins genetics, Arabidopsis Proteins physiology, Flowers physiology, RNA-Binding Proteins metabolism, mRNA Cleavage and Polyadenylation Factors genetics, mRNA Cleavage and Polyadenylation Factors physiology
- Abstract
The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.
- Published
- 2005
- Full Text
- View/download PDF
34. NO flowering.
- Author
-
Simpson GG
- Subjects
- Arabidopsis genetics, Arabidopsis growth & development, Circadian Rhythm, Flowers genetics, Flowers growth & development, Mutation, RNA, Messenger genetics, Arabidopsis physiology, Flowers physiology, Nitric Oxide physiology
- Abstract
The complex control of flowering time ensures that plants flower in conditions favourable for reproductive success. A recent study adds another dimension to this established complexity by revealing that nitric oxide (NO) represses flowering in Arabidopsis. The analysis of recently identified mutants that either overproduce or are compromised in endogenous NO production has identified NO-sensitive features of the circuitry of flowering time control: NO acts to repress the amplification of gene expression dependent on the circadian clock and promotes the accumulation of mRNA encoding a key repressor of flowering, FLC., ((c) 2005 Wiley Periodicals, Inc.)
- Published
- 2005
- Full Text
- View/download PDF
35. Regulated RNA processing in the control of Arabidopsis flowering.
- Author
-
Quesada V, Dean C, and Simpson GG
- Subjects
- Arabidopsis genetics, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Flowers growth & development, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Genes, Plant, MicroRNAs genetics, MicroRNAs metabolism, Models, Biological, RNA Processing, Post-Transcriptional, RNA, Plant genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Arabidopsis growth & development, Arabidopsis metabolism, RNA, Plant metabolism
- Abstract
Flowering time is controlled in order to ensure reproductive success. Molecular genetic analyses in Arabidopsis thaliana have identified many genes regulating this developmental switch. One group of factors which promote flowering do so by down-regulating the expression of the MADS-box floral repressor, FLC. RNA processing appears to play an important role in this regulation as genes within this group encode RNA binding proteins (FCA, FPA and FLK) and an mRNA 3' end processing factor (FY). FCA promotes flowering and negatively autoregulates its own expression post-transcriptionally through a mechanism that involves alternative polyadenylation. FCA physically interacts with FY and this interaction is required for the function FY performs in flowering control and in FCA autoregulation. Potential similarities are emerging in the molecular mechanisms controlling FLC expression and those controlling the floral homeotic gene, AGAMOUS. In addition, microRNAs have been shown to regulate plant developmental processes including the timing to flower. Together, these new data indicate that post-transcriptional regulation of gene expression plays an important role in regulating the floral transition.
- Published
- 2005
- Full Text
- View/download PDF
36. The autonomous pathway: epigenetic and post-transcriptional gene regulation in the control of Arabidopsis flowering time.
- Author
-
Simpson GG
- Subjects
- AGAMOUS Protein, Arabidopsis genetics, AGAMOUS Protein, Arabidopsis metabolism, Arabidopsis genetics, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Down-Regulation, Flowers genetics, Light, MADS Domain Proteins genetics, MADS Domain Proteins metabolism, Signal Transduction, Up-Regulation, Arabidopsis metabolism, Flowers metabolism, Gene Expression Regulation, Plant
- Abstract
Mechanisms that mediate the control of flowering time have been accessed through a molecular genetic approach in Arabidopsis. Flowering is regulated by different pathways and, in the past year, all of the known components of the so-called autonomous pathway have been identified. The autonomous pathway comprises a combination of factors involved in RNA processing and epigenetic regulation that downregulate the floral repressor, FLOWERING LOCUS C (FLC). However, components of the autonomous pathway are more widely conserved in plant species other than Arabidopsis than is FLC. Therefore, the broadest lessons we learn from dissecting the function of the autonomous pathway may be in revealing how precision in regulated gene expression is delivered.
- Published
- 2004
- Full Text
- View/download PDF
37. RNA processing and Arabidopsis flowering time control.
- Author
-
Simpson GG, Quesada V, Henderson IR, Dijkwel PP, Macknight R, and Dean C
- Subjects
- Arabidopsis genetics, Gene Expression Regulation, Plant genetics, Genes, Plant, Arabidopsis physiology, RNA Processing, Post-Transcriptional
- Abstract
Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3'-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.
- Published
- 2004
- Full Text
- View/download PDF
38. Evolution of flowering in response to day length: flipping the CONSTANS switch.
- Author
-
Simpson GG
- Subjects
- Circadian Rhythm, Arabidopsis genetics, Gene Expression Regulation, Plant, Models, Genetic, Oryza genetics, Plants genetics
- Abstract
Day length provides an important environmental cue by signalling conditions favourable for flowering. While Arabidopsis promotes flowering in response to long days, rice promotes flowering in response to short days. Despite this difference, a recent paper reveals that the network controlling this response is highly conserved in these distantly related plants, only the activity of one component is reversed. This reveals how an important developmental process can be diversified for adaptation by using the same set of genes, but regulating them differently., (Copyright 2003 Wiley Periodicals, Inc.)
- Published
- 2003
- Full Text
- View/download PDF
39. Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time.
- Author
-
Quesada V, Macknight R, Dean C, and Simpson GG
- Subjects
- Arabidopsis genetics, Flowers physiology, Genes, Plant, Genes, Reporter, Introns, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA, Plant metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Seedlings anatomy & histology, Seedlings physiology, Transgenes, Arabidopsis physiology, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Gene Expression Regulation, Plant, RNA Processing, Post-Transcriptional, RNA, Messenger metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism
- Abstract
The timing of the transition to flowering is critical for reproductive success in plants. Arabidopsis FCA encodes an RNA-binding protein that promotes flowering. FCA expression is regulated through alternative processing of its pre-mRNA. We demonstrate here that FCA negatively regulates its own expression by ultimately promoting cleavage and polyadenylation within intron 3. This causes the production of a truncated, inactive transcript at the expense of the full-length FCA mRNA, thus limiting the expression of active FCA protein. We show that this negative autoregulation is under developmental control and requires the FCA WW protein interaction domain. Removal of introns from FCA bypasses the autoregulation, and the resulting increased levels of FCA protein overcomes the repression of flowering normally conferred through the up-regulation of FLC by active FRI alleles. The negative autoregulation of FCA may therefore have evolved to limit FCA activity and hence control flowering time.
- Published
- 2003
- Full Text
- View/download PDF
40. FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition.
- Author
-
Simpson GG, Dijkwel PP, Quesada V, Henderson I, and Dean C
- Subjects
- Amino Acid Sequence genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Base Sequence genetics, DNA, Complementary analysis, DNA, Complementary genetics, Flowers metabolism, MADS Domain Proteins genetics, MADS Domain Proteins metabolism, Molecular Sequence Data, Mutation genetics, Plant Proteins genetics, Polyadenylation genetics, Protein Structure, Tertiary genetics, RNA, Messenger genetics, RNA-Binding Proteins genetics, Repressor Proteins genetics, Reproduction genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, mRNA Cleavage and Polyadenylation Factors genetics, mRNA Cleavage and Polyadenylation Factors metabolism, Arabidopsis genetics, Arabidopsis Proteins metabolism, Flowers genetics, Plant Proteins isolation & purification, RNA 3' End Processing genetics, RNA, Messenger metabolism, RNA-Binding Proteins isolation & purification, RNA-Binding Proteins metabolism
- Abstract
The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3' end-processing factor, Pfs2p. FY regulates RNA 3' end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3' end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3' end processing.
- Published
- 2003
- Full Text
- View/download PDF
41. New frontiers in plant development: from genes to phenotype.
- Author
-
Hedden P, Holdsworth MJ, Simpson GG, Edwards KJ, and Lenton JR
- Subjects
- Evolution, Molecular, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Phenotype, Plant Proteins genetics, Plant Proteins physiology, Plants genetics, Reproduction physiology, Signal Transduction genetics, Plant Development, Signal Transduction physiology
- Published
- 2002
- Full Text
- View/download PDF
42. Arabidopsis, the Rosetta stone of flowering time?
- Author
-
Simpson GG and Dean C
- Subjects
- Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis Proteins genetics, Circadian Rhythm, DNA-Binding Proteins genetics, DNA-Binding Proteins physiology, Genes, Plant, Gibberellins metabolism, Meristem physiology, Mutation, Photoperiod, Plant Structures physiology, Seasons, Temperature, Time Factors, Transcription Factors genetics, Transcription Factors physiology, Arabidopsis physiology, Arabidopsis Proteins metabolism, Gene Expression Regulation, Plant
- Abstract
Multiple environmental and endogenous inputs regulate when plants flower. The molecular genetic dissection of flowering time control in Arabidopsis has identified an integrated network of pathways that quantitatively control the timing of this developmental switch. This framework provides the basis to understand the evolution of different reproductive strategies and how floral pathways interact through seasonal progression.
- Published
- 2002
- Full Text
- View/download PDF
43. UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation.
- Author
-
Lambermon MH, Simpson GG, Wieczorek Kirk DA, Hemmings-Mieszczak M, Klahre U, and Filipowicz W
- Subjects
- Amino Acid Sequence, Cloning, Molecular, Gene Expression, Genes, Plant, Heterogeneous-Nuclear Ribonucleoproteins, Introns, Molecular Sequence Data, Nuclear Proteins genetics, Plant Proteins genetics, Plants, Toxic, RNA Splicing, Ribonucleoproteins genetics, Sequence Homology, Amino Acid, Nicotiana genetics, Nicotiana metabolism, Nuclear Proteins metabolism, Plant Proteins metabolism, RNA Precursors metabolism, RNA, Plant metabolism, Ribonucleoproteins metabolism
- Abstract
Efficient splicing of higher plant pre-mRNAs depends on AU- or U-rich sequences in introns. Moreover, AU-rich sequences present in 3'-untranslated regions (3'-UTRs) may play a role in 3' end processing of plant mRNAs. Here, we describe the cloning and characterization of a Nicotiana plumbaginifolia nuclear protein that can be cross-linked to U-rich intron and 3'-UTR sequences in vitro, and associates with nuclear poly(A)(+) RNA in vivo. The protein, UBP1, strongly enhances the splicing of otherwise inefficiently processed introns when overexpressed in protoplasts. It also increases the accumulation of reporter mRNAs that contain suboptimal introns or are intronless. The enhanced accumulation is apparently due to UBP1 interacting with the 3'-UTR and protecting mRNA from exonucleolytic degradation. The effect on mRNA accumulation but not on mRNA splicing was found to be promoter specific. The fact that these effects of UBP1 can be separated suggests that they represent two independent activities. The properties of UBP1 indicate that it is an hnRNP protein that functions at multiple steps to facilitate the nuclear maturation of plant pre-mRNAs.
- Published
- 2000
- Full Text
- View/download PDF
44. Environmental-dependent acceleration of a developmental switch: the floral transition.
- Author
-
Simpson GG and Dean C
- Subjects
- Homeodomain Proteins physiology, Lighting, MADS Domain Proteins, Meristem physiology, Plant Proteins physiology, Plants, Genetically Modified, Transcription Factors physiology, Adaptation, Biological physiology, Arabidopsis physiology, Arabidopsis Proteins, Gene Expression Regulation, Plant physiology
- Abstract
The transition from vegetative growth to reproductive growth in plants in which flowers are produced requires the activation of specific genes. Simpson and Dean discuss two recent reports that characterize the FLOWERING LOCUS T (FT) gene in Arabidopsis, which is part of the floral transition pathway. Unlike many of the known genes that initiate flower production, the FT gene appears to encode a membrane-associated protein that could function in signaling from the cell surface.
- Published
- 2000
- Full Text
- View/download PDF
45. When to switch to flowering.
- Author
-
Simpson GG, Gendall AR, and Dean C
- Subjects
- Genes, Plant, Meristem growth & development, Plant Structures growth & development, Plant Development, Plants genetics
- Abstract
At a certain stage in their life cycle, plants switch from vegetative to reproductive development. This transition is regulated by multiple developmental and environmental cues. These ensure that the plant switches to flowering at a time when sufficient internal resources have been accumulated and the environmental conditions are favorable. The use of a molecular genetic approach in Arabidopsis has resulted in the identification and cloning of many of the genes involved in regulating floral transition. The current view on the molecular function of these genes, their division into different genetic pathways, and how the pathways interact in a complex regulatory network are summarized.
- Published
- 1999
- Full Text
- View/download PDF
46. Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organisation of the spliceosomal machinery.
- Author
-
Simpson GG and Filipowicz W
- Subjects
- Cell Nucleus metabolism, Gene Expression Regulation, Plant, Introns, Nucleic Acid Conformation, RNA Precursors chemistry, RNA, Messenger chemistry, RNA, Plant chemistry, Ribonucleoproteins genetics, Plants genetics, RNA Precursors genetics, RNA Splicing, RNA, Messenger genetics, RNA, Plant genetics, Spliceosomes ultrastructure
- Abstract
The removal of introns from pre-mRNA transcripts and the concomitant ligation of exons is known as pre-mRNA splicing. It is a fundamental aspect of constitutive eukaryotic gene expression and an important level at which gene expression is regulated. The process is governed by multiple cis-acting elements of limited sequence content and particular spatial constraints, and is executed by a dynamic ribonucleoprotein complex termed the spliceosome. The mechanism and regulation of pre-mRNA splicing, and the sub-nuclear organisation of the spliceosomal machinery in higher plants is reviewed here. Heterologous introns are often not processed in higher plants indicating that, although highly conserved, the process of pre-mRNA splicing in plants exhibits significant differences that distinguish it from splicing in yeast and mammals. A fundamental distinguishing feature is the presence of and requirement for AU or U-rich intron sequence in higher-plant pre-mRNA splicing. In this review we document the properties of higher-plant introns and trans-acting spliceosomal components and discuss the means by which these elements combine to determine the accuracy and efficiency of pre-mRNA processing. We also detail examples of how introns can effect regulated gene expression by affecting the nature and abundance of mRNA in plants and list the effects of environmental stresses on splicing. Spliceosomal components exhibit a distinct pattern of organisation in higher-plant nuclei. Effective probes that reveal this pattern have only recently become available, but the domains in which spliceosomal components concentrate were identified in plant nuclei as enigmatic structures some sixty years ago. The organisation of spliceosomal components in plant nuclei is reviewed and these recent observations are unified with previous cytochemical and ultrastructural studies of plant ribonuleoprotein domains.
- Published
- 1996
- Full Text
- View/download PDF
47. Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.
- Author
-
Simpson GG, Clark GP, Rothnie HM, Boelens W, van Venrooij W, and Brown JW
- Subjects
- Amino Acid Sequence, Autoantigens, Base Sequence, Genomic Library, Humans, Molecular Sequence Data, Protein Binding, Protein Biosynthesis, RNA Precursors metabolism, RNA Processing, Post-Transcriptional, RNA, Small Nuclear metabolism, Ribonucleoprotein, U1 Small Nuclear metabolism, Ribonucleoprotein, U2 Small Nuclear metabolism, Ribonucleoproteins, Small Nuclear, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Species Specificity, Transcription, Genetic, snRNP Core Proteins, RNA-Binding Proteins, Ribonucleoprotein, U1 Small Nuclear genetics, Ribonucleoprotein, U2 Small Nuclear genetics, Solanum tuberosum genetics, Spliceosomes
- Abstract
In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.
- Published
- 1995
- Full Text
- View/download PDF
48. The organization of spliceosomal components in the nuclei of higher plants.
- Author
-
Beven AF, Simpson GG, Brown JW, and Shaw PJ
- Subjects
- Base Sequence, Cell Cycle, Hot Temperature, Microscopy, Confocal, Molecular Sequence Data, Oligodeoxyribonucleotides, Pisum sativum metabolism, Ribonucleoproteins, Small Nuclear metabolism, Cell Nucleus ultrastructure, Pisum sativum ultrastructure, Spliceosomes
- Abstract
To analyze the organization of spliceosomal snRNPs in plant nuclei, we have used both immunofluorescence labelling with the antibody 4G3, raised against the human snRNP-specific protein U2B", and in situ hybridization with anti-sense probes to conserved regions of U1, U2 and U6 snRNAs. The organization comprises a fibrous interchromatin network, which may include both interchromatin fibrils and granules, and very prominent nuclear and nucleolar-associated bodies. Double labelling with an anti-p80 coilin antibody shows that these are coiled bodies. Dynamic changes in the labelling pattern were observed through the cell cycle, and in response to and on recovery from heat shock. The similarity of this organization to that observed in mammalian nuclei is strong evidence that it is fundamental to the processing of pre-mRNA in eucaryotes in general.
- Published
- 1995
- Full Text
- View/download PDF
49. Isolation of a maize cDNA encoding a protein with extensive similarity to an inhibitor of protein kinase C and a cyanobacterial open reading frame.
- Author
-
Simpson GG, Clark G, and Brown JW
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Biological Evolution, Cattle, Cyanobacteria genetics, Intracellular Signaling Peptides and Proteins, Molecular Sequence Data, Sequence Alignment, Carrier Proteins, DNA, Complementary genetics, Plant Proteins genetics, Protein Kinase C antagonists & inhibitors, Zea mays genetics
- Abstract
A full-length cDNA clone, Mz2-12, with a predicted amino acid sequence showing extensive similarity to the sequence of a protein inhibitor of protein kinase C, purified from bovine brain, has been isolated from maize. The sequence of Mz2-12 is also similar to an open reading frame of unknown function on a cyanobacterial dicistronic message. The extensive similarity of the three protein sequences identifies a novel class of evolutionarily conserved proteins.
- Published
- 1994
- Full Text
- View/download PDF
50. Plant pre-mRNA splicing and splicing components.
- Author
-
Brown JW, Simpson CG, Simpson GG, Turnbull-Ross AD, and Clark GP
- Subjects
- Base Sequence, DNA genetics, Genetic Vectors, Introns, Molecular Sequence Data, Plant Proteins genetics, Plants, Genetically Modified genetics, Plants, Genetically Modified metabolism, Plants, Toxic, Ribonucleoproteins, Small Nuclear genetics, Solanum tuberosum genetics, Solanum tuberosum metabolism, Spliceosomes metabolism, Nicotiana genetics, Nicotiana metabolism, Plants genetics, Plants metabolism, RNA Precursors genetics, RNA Precursors metabolism, RNA Splicing genetics
- Abstract
Pre-mRNA splicing or the removal of introns from precursor messenger RNAs depends on the accurate recognition of intron sequences by the plant splicing machinery. The major components of this machinery are small nuclear ribonucleoprotein protein particles (snRNPs) which consist of snRNAs and snRNP proteins. We have analysed various aspects of intron sequence and structure in relation to splice site selection and splicing efficiency and we have cloned snRNA genes and a gene encoding the snRNP protein, U2B". In the absence of an in vitro splicing system for plants, transient expression in protoplasts and stable plant transformations have been used to analyse splicing of intron constructs. We aim to address the function of the UsnRNP-specific protein, U2B", via the production of transgenic plants expressing antisense U2B" transcripts and epitope-tagged U2B" protein. In addition, we have cloned genes encoding other proteins which potentially interact with RNA, such as RNA helicases, and strategies involving transgenic plants are being developed to analyse their function.
- Published
- 1993
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.