In vitro and in vivo studies on pre-mRNA splicing in plants

2017 Summer.Includes bibliographical references.To view the abstract, please see the full text of the document

Functional analyses of splice variants of the splicing regulator SR45 in abiotic stresses in Arabidopsis

2013 Summer.Includes bibliographical references.Alternative splicing, a post-transcriptional regulatory mechanism of gene expression, produces multiple mRNAs from a single gene. Alternative splicing increases proteome complexity and regulates gene expression through multiple mechanisms. A number of stresses have been shown to regulate alternative splicing of precursor mRNAs in plants and change transcriptome complexity. Serine/arginine-rich (SR) and SR-like proteins that regulate splicing also undergo extensive alternative splicing in response to various stresses. SR45, an SR-like protein, interacts with several spliceosomal proteins such as U170K, SCL33, U2AF35, and also with an intronic sequence of SR30 and regulates alternative splicing of pre-mRNAs of several other SR genes. It has been previously shown that SR45 pre-mRNA undergoes alternative splicing and produces two alternatively spliced mRNA isoforms (long and short) and the proteins coded by these two isoforms differ in eight amino acids. The two isoforms have distinct biological functions during development where the long isoform is important for flower development while the short isoform is necessary for normal root growth. In this work, I have studied the roles of SR45 and its splice variants in heat and salt tolerance using SR45 mutant (sr45) and transgenic lines complemented with either the long or short isoform. I have found that at different developmental stages sr45 shows high sensitivity to heat stress and salt stress as compared to wild type. The sensitivity of sr45 to heat and salt stresses is rescued by the long isoform but not the short one, suggesting that only the long isoform functions in these stresses. Further molecular analyses have revealed that the relative expression and the splicing pattern of heat shock factors (HSFs), heat shock proteins (HSPs), salt overly sensitive (SOS) genes, ABA signaling pathway genes, and other stress-responsive genes are affected in the sr45 mutant and the long isoform is needed for normal splicing and expression of these genes. Furthermore, an in vitro binding assay showed that SR45 binds to an alternatively spliced intron of HsfA2, suggesting that SR45 directly regulates alternative splicing and expression of HsfA2 under heat stress. In addition to misregulation of expression and splicing of some salt stress responsive genes in the mutant, new splicing isoforms that are affected in the mutant are identified, suggesting the importance of SR45 in fine-tuning gene expression under salt stress. In conclusion, results presented here demonstrate that SR45 functions as a positive regulator of tolerance to two abiotic stresses by modulating the expression and splicing of several stress responsive genes. Further, I show that only the long isoform confers tolerance to these abiotic stresses

Brassinosteroid modulates ethylene synthesis and antioxidant metabolism to protect rice (Oryza sativa) against heat stress-induced inhibition of source‒sink capacity and photosynthetic and growth attributes

This study presents an exploration of the efficacy of brassinosteroids (BRs) and ethylene in mediating heat stress tolerance in rice (Oryza sativa). Heat is one of the major abiotic factors that prominently deteriorates rice production by influencing photosynthetic efficiency, source‒sink capacity, and growth traits. The application of BR (0.5 mM) and ethylene (200 μl l-1) either individually and/or in combination was found to alleviate heat stress-induced toxicity by significantly improving photosynthesis, source‒sink capacity and defense systems; additionally, it reduced the levels of oxidative stress markers and ethylene formation. The study revealed the positive influence of BR in promoting plant growth responses under heat stress through its interplay with ethylene biosynthesis and enhanced plant defense systems. Interestingly, treatment with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG) substantiated that BR application to heat-stressed rice plants enhanced ethylene-dependent pathways to counteract the underlying adversities. Thus, BR action was found to be mediated by ethylene to promote heat tolerance in rice. The present study sheds light on the potential tolerance mechanisms which can ensure rice sustainability under heat stress conditions

The Prevalence of Obesity among School Students and its Relation to Dietary and Physical Habits

BACKGROUND: Recent studies suggest that unhealthy food practices and lack of exercise are the main cause of the progressive increase in the obesity prevalence. AIM: We aim to assess the prevalence of obesity among school students and its relationship to dietary and physical habits. METHODS: A cross-sectional study from 18 intermediate and high schools located in Taif, Saudi Arabia, between April 2014 and June 2015. We excluded any student with a psychological disease or chronic diseases. We calculated the body mass index (BMI) of students by measuring their height and weight. Dietary, physical, sleep habits, and socioeconomic status were assessed. RESULTS: A total of 2943 students participated with a mean age of 15.4 ± 1.7 years. The majority of these were boys and attending government schools with a mean BMI 22.7 ± 8.6 kg/m2. Overall, 13.6% were overweight and 12.8% were obese. One-third of students report a sedentary lifestyle. Compared to girls, boys were more likely to have lower BMI (p < 0.001), be overweight (p < 0.001), exercise for >300 min/week (p < 0.001), own electronic devices other than smartphone (p < 0.001), use the electronic devices <3 h/day (p = 0.007), eat their meals with their family (p < 0.001), eat fruits and vegetables daily (p < 0.05), exercise for >300 min/week (p < 0.001), and walk to and from school at least once weekly (p < 0.001). CONCLUSION: A quarter of the students were either overweight or obese. Girls were more likely to be obese with less favorable socioeconomic, media consumption, dietary, and physical profiles

Heat Stroke in Emergency Department: Diagnosis and Management

Background: Heat stroke is a severe health concern with the potential for multi-organ failure, necessitating rapid and effective management. With rising global temperatures, there is increasing concern regarding the vulnerability of populations in high-heat areas, notably in Saudi Arabia, especially during the annual Hajj pilgrimage. Objective: This paper aims to review the epidemiology, evaluation and management techniques of heat stroke, emphasizing the situation during Hajj pilgrimages in Saudi Arabia, and to outline the best practices for emergency management. Methodology: A comprehensive review of literature and studies related to heat stroke, both globally and specific to Saudi Arabia, was undertaken. An in-depth analysis of emergency management, including initial assessment, cooling methods, organ support, medication, and prevention strategies, was conducted. Results: Heat stroke remains a significant cause of emergency department visits, with specific groups, such as men and the elderly, being more susceptible. During the Hajj in 2016, 267 patients were diagnosed with heat-related illnesses, with heatstroke accounting for 29% of these cases. With the threat of global warming, studies indicate a potential tenfold increase in heat stroke risk with a 2°C rise in temperatures. Swift and comprehensive cooling is pivotal for recovery. Management emphasizes rapid recognition, assessment, and varied cooling methods, along with targeted treatments for organ dysfunctions. Prevention strategies play a vital role, given the higher efficacy and practicality over treating organ dysfunctions. Conclusion: Heat stroke is a pressing health challenge, particularly in high-risk environments like Saudi Arabia during the Hajj pilgrimage. While effective emergency management protocols exist, an emphasis on prevention is crucial. It is imperative to incorporate a comprehensive approach to address both the immediate threat and long-term risks of heat stroke, especially with the looming challenge of global warming

The Splicing Factor SR45 Negatively Regulates Anthocyanin Accumulation under High-Light Stress in <i>Arabidopsis thaliana</i>

High-intensity light (HL) greatly induces the accumulation of anthocyanin, a fundamental compound in photoprotection and antioxidation. Many mechanisms regulating anthocyanin biosynthesis are well-characterized across developmental and environmental conditions; however, post-transcriptional regulation of its biosynthesis remains unclear. RNA splicing is one mechanism of post-transcriptional control and reprogramming in response to different developmental cues and stress conditions. The Arabidopsis splicing modulator SR45 regulates a number of developmental and environmental stress responses. Here, we investigated the role of SR45 and its isoforms in HL-induced anthocyanin accumulation. We found that the SR45 promoter contains light-responsive cis-elements, and that light stress significantly increases SR45 expression. Furthermore, we found that mutant plants lacking SR45 function (sr45) accumulate significantly more anthocyanin under HL. SR45 is alternatively spliced to produce two proteins, SR45.1 and SR45.2, which differ by seven amino acids. Intriguingly, these isoforms exhibited distinct functions, with only SR45.1 reversing anthocyanin accumulation in the sr45 plants. We also identified possible SR45 target genes that are involved in anthocyanin synthesis. Consistent with the antioxidant role of anthocyanin, we found that sr45 mutants and SR45.2 overexpression lines accumulate anthocyanin and better tolerate paraquat which induces oxidative stress. Collectively, our results reveal that the Arabidopsis splicing regulator SR45 inhibits anthocyanin accumulation under HL, which may negatively affect oxidative stress tolerance. This study illuminates splicing-level regulation of anthocyanin production in response to light stress and offers a possible target for genetic modification to increase plant stress tolerance

Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

Abstract Background Pre-mRNA splicing is an essential post-transcriptional process in all eukaryotes. In vitro splicing systems using nuclear or cytoplasmic extracts from mammalian cells, yeast, and Drosophila have provided a wealth of mechanistic insights into assembly and composition of the spliceosome, splicing regulatory proteins and mechanisms of pre-mRNA splicing in non-plant systems. The lack of an in vitro splicing system prepared from plant cells has been a major limitation in splicing research in plants. Results Here we report an in vitro splicing assay system using plant nuclear extract. Several lines of evidence indicate that nuclear extract derived from Arabidopsis seedlings can convert pre-mRNA substrate (LHCB3) into a spliced product. These include: (1) generation of an RNA product that corresponds to the size of expected mRNA, (2) a junction-mapping assay using S1 nuclease revealed that the two exons are spliced together, (3) the reaction conditions are similar to those found with non-plant extracts and (4) finally mutations in conserved donor and acceptor sites abolished the production of the spliced product. Conclusions This first report on the plant in vitro splicing assay opens new avenues to investigate plant spliceosome assembly and composition, and splicing regulatory mechanisms specific to plants

MOESM4 of Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

Additional file 4: Figure S4. (A) A schematic diagram other Arabidopsis pre-mRNA substrates that were tested for in vitro splicing. Gene names and ID numbers are displayed at the top of each substrate. Introns are black thin lines, first exons are orange boxes, and second exons are green boxes. SP6 promoters are red boxes. Numbers within the boxes or at the top of the introns indicate the length (in nucleotides (nt)) of each region. Introns’ UA content is shown under each intron. The numbers listed on the right indicate the full length of each substrate. The length of exons and introns is drawn to scale

MOESM6 of Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

Additional file 6: Figure S5. In vitro splicing assay of different Arabidopsis [32P] labeled pre-mRNA substrates. Radiolabelled AP2M, TIP41 and PTB1 pre-mRNA substrates were incubated individually at 30 °C in the Arabidopsis nuclear extract as described previously. Samples were withdrawn at indicated times, extracted [32P]-RNA and analyzed by electrophoresis on a 6% polyacrylamide-7 M urea gel, followed by autoradiography. The sizes of the pre-mRNA substrates are indicated at right

MOESM5 of Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

Additional file 5: Figure S4. (B) Sequences of the other Arabidopsis pre-mRNA substrates tested for in vitro splicing. Sequences were extracted from The Arabidopsis Information Resource (TAIR). Exons are shown in upper case letters, while the introns are shown in lower case. SP6 promoter sequence is highlighted in yellow; primers are highlighted in green, and conserved splicing sites are highlighted in black