Temperature-dependent Alternative Splicing Research Articles

Thermoregulated transcriptomics: the molecular basis and biological significance of temperature-dependent alternative splicing.

Temperature-dependent alternative splicing (AS) is a crucial mechanism for organisms to adapt to varying environmental temperatures. In mammals, even slight fluctuations in body temperature are sufficient to drive significant AS changes in a concerted manner. This dynamic regulation allows organisms to finely tune gene expression and protein isoform diversity in response to temperature cues, ensuring proper cellular function and physiological adaptation. Understanding the molecular mechanisms underlying temperature-dependent AS thus provides valuable insights into the intricate interplay between environmental stimuli and gene expression regulation. In this review, we provide an overview of recent advances in understanding temperature-regulated AS across various biological processes and systems. We will discuss the machinery sensing and translating temperature cues into changed AS patterns, the adaptation of the splicing regulatory machinery to extreme temperatures, the role of temperature-dependent AS in shaping the transcriptome, functional implications and the development of potential therapeutics targeting temperature-sensitive AS pathways.

N-terminal acetylation orchestrates glycolate-mediated ROS homeostasis to promote rice thermoresponsive growth.

Climate warming poses a significant threat to global crop production and food security. However, our understanding of the molecular mechanisms governing thermoresponsive development in crops remains limited. Here we report that the auxiliary subunit of N-terminal acetyltransferase A (NatA) in rice OsNAA15 is a prerequisite for rice thermoresponsive growth. OsNAA15 produces two isoforms OsNAA15.1 and OsNAA15.2, via temperature-dependent alternative splicing. Among the two, OsNAA15.1 is more likely to form a stable and functional NatA complex with the potential catalytic subunit OsNAA10, leading to a thermoresponsive N-terminal acetylome. Intriguingly, while OsNAA15.1 promotes plant growth under elevated temperatures, OsNAA15.2 exhibits an inhibitory effect. We identified two glycolate oxidases (GLO1/5) as major substrates from the thermoresponsive acetylome. These enzymes are involved in hydrogen peroxide (H2O2) biosynthesis via glycolate oxidation. N-terminally acetylated GLO1/5 undergo their degradation through the ubiquitin-proteasome system. This leads to reduced reactive oxygen species (ROS) production, thereby promoting plant growth, particularly under high ambient temperatures. Conclusively, our findings highlight the pivotal role of N-terminal acetylation in orchestrating the glycolate-mediated ROS homeostasis to facilitate thermoresponsive growth in rice.

Structural analysis of temperature-dependent alternative splicing of HsfA2 pre-mRNA from tomato plants

ABSTRACT Temperature-dependent alternative splicing was recently demonstrated for intron 2 of the gene coding for heat shock factor HsfA2 of the tomato plant Solanum lycopersicum, but the molecular mechanism regulating the abundance of such temperature-dependent splice variants is still unknown. We report here on regulatory pre-mRNA structures that could function as regulators by controlling the use of splice sites in a temperature-dependent manner. We investigate pre-mRNA structures at the splice sites of intron 2 of the gene coding for HsfA2 from S. lycopersicum using NMR- and CD-spectroscopy as well as in-line probing. The pre-mRNA undergoes conformational changes between two different secondary structures at the 3ʹ splice site of the intron in a temperature-dependent manner. Previously, it was shown that three single nucleotide polymorphisms (SNPs) in intron 2 of the HsfA2 pre-mRNA affect the splicing efficiency of its pre-mRNA and are linked to the thermotolerance in different tomato species. By comparing pre-mRNA fragments of the tomato species S. lycopersicum and S. peruvianum, we show that these SNPs result in substantial structural differences between the pre-mRNAs of the two species.

Insights into the role of alternative splicing in plant temperature response.

Alternative splicing occurs in all eukaryotic organisms. Since the first description of multiexon genes and the splicing machinery, the field has expanded rapidly, especially in animals and yeast. However, our knowledge about splicing in plants is still quite fragmented. Though eukaryotes show some similarity in the composition and dynamics of the splicing machinery, observations of unique plant traits are only starting to emerge. For instance, plant alternative splicing is closely linked to their ability to perceive various environmental stimuli. Due to their sessile lifestyle, temperature is a central source of information allowing plants to adjust their development to match current growth conditions. Hence, seasonal temperature fluctuations and day-night cycles can strongly influence plant morphology across developmental stages. Here we discuss the available data about temperature-dependent alternative splicing in plants. Given its fragmented state it is not always possible to fit specific observations into a coherent picture, yet it is sufficient to estimate the complexity of this field and the need of further research. Better understanding of alternative splicing as a part of plant temperature response and adaptation may also prove to be a powerful tool for both, fundamental and applied sciences.

Temperature-Dependent Alternative Splicing of Precursor mRNAs and Its Biological Significance: A Review Focused on Post-Transcriptional Regulation of a Cold Shock Protein Gene in Hibernating Mammals.

Multiple mRNA isoforms are often generated during processing such as alternative splicing of precursor mRNAs (pre-mRNA), resulting in a diversity of generated proteins. Alternative splicing is an essential mechanism for the functional complexity of eukaryotes. Temperature, which is involved in all life activities at various levels, is one of regulatory factors for controlling patterns of alternative splicing. Temperature-dependent alternative splicing is associated with various phenotypes such as flowering and circadian clock in plants and sex determination in poikilothermic animals. In some specific situations, temperature-dependent alternative splicing can be evoked even in homothermal animals. For example, the splicing pattern of mRNA for a cold shock protein, cold-inducible RNA-binding protein (CIRP or CIRBP), is changed in response to a marked drop in body temperature during hibernation of hamsters. In this review, we describe the current knowledge about mechanisms and functions of temperature-dependent alternative splicing in plants and animals. Then we discuss the physiological significance of hypothermia-induced alternative splicing of a cold shock protein gene in hibernating and non-hibernating animals.

A Conserved Kinase-Based Body-Temperature Sensor Globally Controls Alternative Splicing and Gene Expression

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins invitro and invivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

A Conserved Kinase-Based Body Temperature Sensor Globally Controls Alternative Splicing and Gene Expression

Homoeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remains elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation loop. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR-proteins in vitro and in vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

Contribution of major FLM isoforms to temperature-dependent flowering in Arabidopsis thaliana.

FLOWERING LOCUS M (FLM), a component of the thermosensory flowering time pathway in Arabidopsis thaliana, is regulated by temperature-dependent alternative splicing (AS). The main splicing variant, FLM-β, is a well-documented floral repressor that is down-regulated in response to increasing ambient growth temperature. Two hypotheses have been formulated to explain how flowering time is modulated by AS of FLM. In the first model a second splice variant, FLM-δ, acts as a dominant negative isoform that competes with FLM-β at elevated ambient temperatures, thereby indirectly promoting flowering. Alternatively, it has been suggested that the induction of flowering at elevated temperatures is caused only by reduced FLM-β expression. To better understand the role of the two FLM splice forms, we employed CRISPR/Cas9 technology to specifically delete the exons that characterize each splice variant. Lines that produced repressive FLM-β but were incapable of producing FLM-δ were late flowering. In contrast, FLM-β knockout lines that still produced FLM-δ flowered early, but not earlier than the flm-3 loss of function mutant, as would be expected if FLM-δ had a dominant-negative effect on flowering. Our data support the role of FLM-β as a flower repressor and provide evidence that a contribution of FLM-δ to the regulation of flowering time in wild-type A. thaliana seems unlikely.

Chromatin marks and ambient temperature-dependent flowering strike up a novel liaison

A distinct chromatin mark, H3K36me3, has been found to engage in temperature-dependent alternative splicing and ambient temperature-dependent flowering-time control in Arabidopsis.

The Arabidopsis sickle Mutant Exhibits Altered Circadian Clock Responses to Cool Temperatures and Temperature-Dependent Alternative Splicing

The circadian clock allows plants to anticipate and respond to daily changes in ambient temperature. Mechanisms establishing the timing of circadian rhythms in Arabidopsis thaliana through temperature entrainment remain unclear. Also incompletely understood is the temperature compensation mechanism that maintains consistent period length within a range of ambient temperatures. A genetic screen for Arabidopsis mutants affecting temperature regulation of the PSEUDO-RESPONSE REGULATOR7 promoter yielded a novel allele of the SICKLE (SIC) gene. This mutant, sic-3, and the existing sic-1 mutant both exhibit low-amplitude or arrhythmic expression of core circadian clock genes under cool ambient temperature cycles, but not under light-dark entrainment. sic mutants also lengthen free running period in a manner consistent with impaired temperature compensation. sic mutant alleles accumulate LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) splice variants, among other alternatively spliced transcripts, which is exacerbated by cool temperatures. The cca1-1 lhy-20 double mutant is epistatic to sic-3, indicating the LHY and CCA1 splice variants are needed for sic-3 circadian clock phenotypes. It is not expected that SIC is directly involved in the circadian clock mechanism; instead, SIC likely contributes to pre-mRNA metabolism, and the splice variants that accumulate in sic mutants likely affect the circadian clock response to cool ambient temperature.

MAF2 Is Regulated by Temperature-Dependent Splicing and Represses Flowering at Low Temperatures in Parallel with FLM.

Plants enter their reproductive phase when the environmental conditions are favourable for the successful production of progeny. The transition from vegetative to reproductive phase is influenced by several environmental factors including ambient temperature. In the model plant Arabidopsis thaliana, SHORT VEGETATIVE PHASE (SVP) is critical for this pathway; svp mutants cannot modify their flowering time in response to ambient temperature. SVP encodes a MADS-box transcription factor that directly represses genes that promote flowering. SVP binds DNA in complexes with other MADS-box transcription factors, including FLOWERING LOCUS M (FLM), which acts with SVP to repress the floral transition at low temperatures. Small temperature changes post-transcriptionally regulate FLM through temperature-dependent alternative splicing (TD-AS). As ambient temperature increases, the predominant FLM splice isoform shifts to encode a protein incapable of exerting a repressive effect on flowering. Here we characterize a closely related MADS-box transcription factor, MADS AFFECTING FLOWERING2 (MAF2), which has independently evolved TD-AS. At low temperatures the most abundant MAF2 splice variant encodes a protein that interacts with SVP to repress flowering. At increased temperature the relative abundance of splice isoforms shifts in favour of an intron-retaining variant that introduces a premature termination codon. We show that this isoform encodes a protein that cannot interact with SVP or repress flowering. At lower temperatures MAF2 and SVP repress flowering in parallel with FLM and SVP, providing an additional input to sense ambient temperature for the control of flowering.

Temperature-dependent regulation of flowering by antagonistic FLM variants

The appropriate timing of flowering is crucial for plant reproductive success. It is therefore not surprising that intricate genetic networks have evolved to perceive and integrate both endogenous and environmental signals, such as carbohydrate and hormonal status, photoperiod and temperature. In contrast to our detailed understanding of the vernalization pathway, little is known about how flowering time is controlled in response to changes in the ambient growth temperature. In Arabidopsis thaliana, the MADS-box transcription factor genes FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) have key roles in this process. FLM is subject to temperature-dependent alternative splicing. Here we report that the two main FLM protein splice variants, FLM-β and FLM-δ, compete for interaction with the floral repressor SVP. The SVP-FLM-β complex is predominately formed at low temperatures and prevents precocious flowering. By contrast, the competing SVP-FLM-δ complex is impaired in DNA binding and acts as a dominant-negative activator of flowering at higher temperatures. Our results show a new mechanism that controls the timing of the floral transition in response to changes in ambient temperature. A better understanding of how temperature controls the molecular mechanisms of flowering will be important to cope with current changes in global climate.

The NH2-terminal Domain of the Chloroplast GrpE Homolog CGE1 Is Required for Dimerization and Cochaperone Function in Vivo

GrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s. We have previously identified a chloroplast homolog of GrpE in Chlamydomonas reinhardtii, termed CGE1. CGE1 exists as two isoforms, CGE1a and CGE1b, which are generated by temperature-dependent alternative splicing. CGE1b contains additional valine and glutamine residues in its extreme NH2-terminal region. Here we show that CGE1a is predominant at lower temperatures but that CGE1b becomes as abundant as CGE1a at elevated temperatures. Coimmunoprecipitation experiments revealed that CGE1b had a approximately 25% higher affinity for its chloroplast chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended alpha-helix formed by GrpE NH2 termini is 34 amino acids longer in CGE1 than in Escherichia coli GrpE and appears to contain a coiled coil motif. Progressive deletions of this coiled coil increasingly impaired the ability of CGE1 to form dimers, to interact with DnaK at elevated temperatures, and to complement temperature-sensitive growth of a DeltagrpE E. coli strain. In contrast, deletion of the four-helix bundle required for dimerization of E. coli GrpE did not affect CGE1 dimer formation. Circular dichroism measurements revealed that CGE1, like GrpE, undergoes two thermal transitions, the first of which is in the physiologically relevant temperature range (midpoint approximately 45 degrees C). Truncating the NH2-terminal coiled coil shifted the second transition to lower temperatures, whereas removal of the four-helix bundle abolished the first transition. Our data suggest that bacterial GrpE and chloroplast CGE1 share similar structural and biochemical properties, but some of these, like dimerization, are realized by different domains.