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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

BIN1 protein isoforms are differentially expressed in astrocytes, neurons, and microglia: neuronal and astrocyte BIN1 are implicated in tau pathology

Background: Identified as an Alzheimer’s disease (AD) susceptibility gene by genome wide-association studies, BIN1 has 10 isoforms that are expressed in the Central Nervous System (CNS). The distribution of these isoforms in different cell types, as well as their role in AD pathology still remains unclear. Methods: Utilizing antibodies targeting specific BIN1 epitopes in human post-mortem tissue and analyzing mRNA expression data from purified microglia, we identified three isoforms expressed in neurons and astrocytes (isoforms 1, 2 and 3) and four isoforms expressed in microglia (isoforms 6, 9, 10 and 12). The abundance of selected peptides, which correspond to groups of BIN1 protein isoforms, was measured in dorsolateral prefrontal cortex, and their relation to neuropathological features of AD was assessed. Results: Peptides contained in exon 7 of BIN1’s N-BAR domain were found to be significantly associated with ADrelated traits and, particularly, tau tangles. Decreased expression of BIN1 isoforms containing exon 7 is associated with greater accumulation of tangles and subsequent cognitive decline, with astrocytic rather than neuronal BIN1 being the more likely culprit. These effects are independent of the BIN1 AD risk variant. Conclusions: Exploring the molecular mechanisms of specific BIN1 isoforms expressed by astrocytes may open new avenues for modulating the accumulation of Tau pathology in AD. Keywords: BIN1 isoforms, Alzheimer’s disease, Amyloid, Tau, Microglia, Astrocytes, Neurons

Taga, Mariko↗
DOE OSTI2025

Identification and Characterization of Lipid Droplet-Associated Protein (LDAP) Isoforms from Tung Tree (Vernicia fordii)

Lipid droplets (LDs) are cytoplasmic organelles responsible primarily for the storage of neutral lipids, such as triacyclglycerols (TAGs). Derived from the endoplasmic reticulum bilayer, LDs are composed of a hydrophobic lipid core encased by a phospholipid monolayer and surface-associated proteins. To date, only a relatively few LD ‘coat’ proteins in plants have been identified and characterized, most of which come from studies of the model plant Arabidopsis thaliana. To expand our knowledge of the plant LD proteome, the LD-associated protein (LDAP) family from the tung tree (Vernicia fordii), whose seeds are rich in a commercially valuable TAG containing the conjugated fatty acid α-eleostearic acid (C18:3Δ9cis,11trans,13trans [α-ESA]), was identified and characterized. Based on the tung tree transcriptome, three LDAP isoforms (VfLDAP1-3) were elucidated and the encoded proteins distinctly clustered into three clades along with their respective isoforms from other angiosperm species. Ectopic expression of the VfLDAPs in Nicotiana benthamiana leaves revealed that they localized specifically to LDs and influenced LD numbers and sizes, as well as increasing TAG content and altering TAG fatty acid composition. Interestingly, in a partially reconstructed TAG-ESA biosynthetic pathway, the co-expression of VfLDAP3 and, to a lesser degree, VfLDAP2, significantly increased the content of α-ESA stored within the LDs. These results suggest that the VfLDAPs can influence the steady-state content and composition of TAG in plant cells and that certain LDAP isoforms may have evolved to more efficiently package TAGs into LDs containing unusual fatty acids, such as α-ESA.

Plant Sciences↗
DOE OSTI2025

Dynamical Signatures of Thermotoga maritima Maltose-Binding Proteins Affected by Ligand Binding

Functional segregation among protein isoforms depends on the interplay of their overall structures and the molecular dynamics of these structures. Thermotoga maritima maltose-binding protein (tmMBP) isoforms show size-dependent differential binding of maltose and malto-oligomers while maintaining remarkable fold conservation. This differential behavior needs detailed characterization in native-like aqueous conditions to understand the effects of protein dynamics on ligand binding and recognition. Small-angle neutron scattering (SANS), neutron spin echo (NSE) spectroscopy, and dynamic light scattering (DLS) were used in conjunction with previously published computational molecular dynamics (MD) simulations to understand the dynamic behavior of tmMBPs experimentally. SANS provided information on the overall structure of the molecules, while NSE was used to determine the dynamics in the nanosecond time scale. Both tmMBP2 and tmMBP3 have a bidomain architecture linked with a flexible hinge, with the binding pocket sitting in the cleft between the two domains. tmMBP2 and tmMBP3 showed different solution dynamics, with the translational and rotational components dominating the dynamics of both systems, resulting in a clear differentiation of their diffusion pattern. A faster dynamics component was also observed and was attributed to segmental dynamics. Differences observed between the ligand-free (apo) and ligand-bound (holo) states of the two proteins are attributed to conformational entropy. Our results highlight the intricacies of how structure and dynamics can together shape binding to a repertoire of substrates in structurally similar proteins.

Diffusion↗
DOE OSTI2022

Photosynthetic modulation during the diurnal cycle in a unicellular diazotrophic cyanobacterium grown under nitrogen-replete and nitrogen-fixing conditions

Abstract Cyanobacteria are the only oxygenic photosynthetic organisms that can fix nitrogen. In diazotrophic cyanobacteria, the regulation of photosynthesis during the diurnal cycle is hypothesized to be linked with nitrogen fixation and involve the D1 protein isoform PsbA4. The amount of bioavailable nitrogen has a major impact on productivity in aqueous environments. In contrast to low- or nitrogen-fixing (−N) conditions, little data on photosynthetic regulation under nitrogen-replete (+ N) conditions are available. We compared the regulation of photosynthesis under −N and + N conditions during the diurnal cycle in wild type and a psbA4 deletion strain of the unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. We observed common changes to light harvesting and photosynthetic electron transport during the dark in + N and −N conditions and found that these modifications occur in both diazotrophic and non-diazotrophic cyanobacteria. Nitrogen availability increased PSII titer when cells transitioned from dark to light and promoted growth. Under −N conditions, deletion of PsbA4 modified charge recombination in dark and regulation of PSII titer during dark to light transition. We conclude that darkness impacts the acceptor-side modifications to PSII and photosynthetic electron transport in cyanobacteria independently of the nitrogen-fixing status and the presence of PsbA4.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

RNA Splicing Events in Circulation Distinguish Individuals With and Without New-onset Type 1 Diabetes

Context: Alterations in RNA splicing may influence protein isoform diversity that contributes to or reflects the pathophysiology of certain diseases. Whereas specific RNA splicing events in pancreatic islets have been investigated in models of inflammation in vitro, how RNA splicing in the circulation correlates with or is reflective of type 1 diabetes (T1D) disease pathophysiology in humans remains unexplored. Objective: To use machine learning to investigate if alternative RNA splicing events differ between individuals with and without new-onset T1D and to determine if these splicing events provide insight into T1D pathophysiology. Methods: RNA deep sequencing was performed on whole blood samples from 2 independent cohorts: a training cohort consisting of 12 individuals with new-onset T1D and 12 age- and sex-matched nondiabetic controls and a validation cohort of the same size and demographics. Machine learning analysis was used to identify specific isoforms that could distinguish individuals with T1D from controls. Results: Distinct patterns of RNA splicing differentiated participants with T1D from unaffected controls. Notably, certain splicing events, particularly involving retained introns, showed significant association with T1D. Machine learning analysis using these splicing events as features from the training cohort demonstrated high accuracy in distinguishing between T1D subjects and controls in the validation cohort. Gene Ontology pathway enrichment analysis of the retained intron category showed evidence for a systemic viral response in T1D subjects. Conclusion: Alternative RNA splicing events in whole blood are significantly enriched in individuals with new-onset T1D and can effectively distinguish these individuals from unaffected controls. Further, our findings also suggest that RNA splicing profiles offer the potential to provide insights into disease pathogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Genome-Wide Identification of Splicing Quantitative Trait Loci (sQTLs) in Diverse Ecotypes of Arabidopsis thaliana

Alternative splicing (AS) of pre-mRNAs contributes to transcriptome diversity and enables plants to generate different protein isoforms from a single gene and/or fine-tune gene expression during different development stages and environmental changes. Although AS is pervasive, the genetic basis for differential isoform usage in plants is still emerging. In this study, we performed genome-wide analysis in 666 geographically distributed diverse ecotypes of Arabidopsis thaliana to identify genomic regions [splicing quantitative trait loci (sQTLs)] that may regulate differential AS. These ecotypes belong to different microclimatic conditions and are part of the relict and non-relict populations. Although sQTLs were spread across the genome, we observed enrichment for trans-s QTL ( trans-s QTLs hotspots) on chromosome one. Furthermore, we identified several sQTL (911) that co-localized with trait-linked single nucleotide polymorphisms (SNP) identified in the Arabidopsis genome-wide association studies (AraGWAS). Many sQTLs were enriched among circadian clock, flowering, and stress-responsive genes, suggesting a role for differential isoform usage in regulating these important processes in diverse ecotypes of Arabidopsis. In conclusion, the current study provides a deep insight into SNPs affecting isoform ratios/genes and facilitates a better mechanistic understanding of trait-associated SNPs in GWAS studies. To the best of our knowledge, this is the first report of sQTL analysis in a large set of Arabidopsis ecotypes and can be used as a reference to perform sQTL analysis in the Brassicaceae family. Since whole genome and transcriptome datasets are available for these diverse ecotypes, it could serve as a powerful resource for the biological interpretation of trait-associated loci, splice isoform ratios, and their phenotypic consequences to help produce more resilient and high yield crop varieties.

Arabidopsis thaliana↗
DOE OSTI2021

Isoform-specific functions of synaptopodin-2 variants in cytoskeleton stabilization and autophagy regulation in muscle under mechanical stress

Highlights: • Muscle cells express different synaptopodin-2 isoforms with individual functions. • Reduced SYNPO2 expression results in enhanced vulnerability of myofibrils. • SYNPO2e recruites BAG3 to mechanically stressed myofibrillar areas. • SYNPO2 is an organizer and regulator of BAG3-containing proteostasis systems. • BAG3 forms functionally distinct protein complexes with different SYNPO2 isoforms. Protein homeostasis (proteostasis) in multicellular organisms depends on the maintenance of force-bearing and force-generating cellular structures. Within myofibrillar Z-discs of striated muscle, isoforms of synaptopodin-2 (SYNPO2/myopodin) act as adapter proteins that are engaged in proteostasis of the actin-crosslinking protein filamin C (FLNc) under mechanical stress. SYNPO2 directly binds F-actin, FLNc and α-actinin and thus contributes to the architectural features of the actin cytoskeleton. By its association with autophagy mediating proteins, i.e. BAG3 and VPS18, SYNPO2 is also engaged in protein quality control and helps to target mechanical unfolded and damaged FLNc for degradation. Here we show that deficiency of all SYNPO2-isoforms in myotubes leads to decreased myofibrillar stability and deregulated autophagy under mechanical stress. In addition, isoform-specific proteostasis functions were revealed. The PDZ-domain containing variant SYNPO2b and the shorter, PDZ-less isoform SYNPO2e both localize to Z-discs. Yet, SYNPO2e is less stably associated with the Z-disc than SYNPO2b, and is dynamically transferred into FLNc-containing myofibrillar lesions under mechanical stress. SYNPO2e also recruits BAG3 into these lesions via interaction with the WW domain of BAG3. Our data provide evidence for a role of myofibrillar lesions as a transient quality control compartment essential to prevent and repair contraction-induced myofibril damage in muscle and indicate an important coordinating activity for SYNPO2 therein.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

GENE-43. Targeting GABPb1L Inhibits In Vivo Growth of TERT Promoter Mutant Glioblastoma

Understanding cancer cell immortality in primary glioblastoma (GBM) is essential for the development of more informed treatments. Multiple cancer types, including >80% of GBMs, undergo immortalization by reactivating Telomerase Reverse Transcriptase (TERT) through acquired mutations in the TERT promoter. TERT, the catalytically active and rate-limiting subunit of telomerase, functions to maintain telomeres, which cap and protect the ends of chromosomes. Our previous work has demonstrated that the transcription factor GABP - and specifically its tetramer-forming isoform GABPb1L - binds and activates the mutant TERT promoter. The generation of CRISPR-induced indels in GABPb1L results in a gradual loss of cell viability in TERT promoter mutant but not TERT promoter wild type tumor cells in vitro, but the extent to which GABPb1L function is compromised in this setting is unclear. Thus, the potential for use of GABPb1L as an effective therapeutic target for TERT promoter mutant GBM requires further investigation. Here, we use CRISPR-based strategies to demonstrate that full knockout of GABPb1L is rapidly lethal in TERT promoter mutant cells in vitro, in association with a decrease in both TERT mRNA and telomerase activity. Heterozygous deletion of GABPb1L in the context of TERT promoter mutations leads to slowed growth of orthotopic xenograft tumors in mice, and prolonged survival. Additionally, inducible RNAi-mediated inhibition of GABPb1L in growing tumors is also capable of decreasing tumor burden and increasing survival, further strongly suggesting that targeting GABPb1L in patient tumors could be a viable treatment strategy. Finally, reduced GABPb1L synergizes with temozolomide (TMZ) therapy such that TMZ treatment in the context of low GABPb1L and low TERT leads to a complete ablation of orthotopic GBM xenografts. These results highlight the potential to improve disease outcomes by targeting TERT through inhibition of GABPb1L, particularly in conjunction with TMZ treatment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms

Abstract Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana , to rescue the collapsed vessels, short stems, and low cellulose content of the irx3‐1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate “gating loop.” A low‐to‐high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue‐specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella ) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

Burris, Jason N.↗
DOE OSTI2019

eIFiso4G Augments the Synthesis of Specific Plant Proteins Involved in Normal Chloroplast Function

The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis ( Arabidopsis thaliana )—genes encoding the cap binding protein eIFiso4E ( eifiso4e ) and two isoforms of the large subunit scaffolding protein eIFiso4G ( i4g1 and i4g2 ). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants ( i4g1 , i4g2 , i4e , i4g1 x i4g2 , and i4g1 x i4g2 x i4e [ i4f ]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 x i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Large-scale survey of prion protein genetic variability in scrapie disease-free goats from the United States

Scrapie is a slowly progressive neurodegenerative disease of small ruminants caused by an accumulation of an abnormal isoform of prion protein in the central nervous system. Polymorphisms of the prion protein gene (PRNP) strongly modulate scrapie resistance and incubation period in goats. The aim of this study was to identify PRNP genetic variability in goats across the United States. Blood from a total of 6,029 apparent scrapie disease-free goats from 654 operations and 19 breeds were analyzed. Sequencing of PRNP revealed 26 genotypes with different rates based on eight codons. The GG127, RR154, and QQ222 genotypes were predominant and showed a remarkably high rate across all goats. The QK222 and NS146 genotypes, known to be protective against scrapie, were found in 0.6% [with 95% CI = (0.3, 1.2)] and 22.0% [95% CI = (19.1, 25.2)] of goats, respectively. The QK222 genotype was found in 23.1% of Oberhasli goats tested, with 95%CI = (3.9, 68.7)] and 22.0% of Toggenburg goats tested with 95%CI = (9.7, 42.5)], while NS146 was found in 65.5% of Savannah goats tested, with 95%CI = (30.8, 89.9), 36.7% of Boer goats tested, with 95%CI = (33.1, 40.4), 36.3% of Nubian goats tested, with 95%CI = (27.0, 46.7)], and 35.6% of LaMancha goats tested, with 95%CI = (22.8, 50.8%). The MM142 and IM142 genotypes were found more frequently in goats on dairy operations, while the HR143, NS146, and ND146 genotypes were found more frequently in goats on meat operations. Goats in the east region had a higher percentage of goats with RH154, RQ211, and QK222 genotypes than goats in the west region. The results of this study showed high genetic variability of PRNP among the U.S. goat population, with differences by location and breed, and may serve as a rationale for development of goat breeding programs at the national level to mitigate the risk of scrapie.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

GRIK phosphorylates and activates KIN10 which also promotes its degradation

The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (a) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to low sugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Alternative Splicing and Protein Diversity: Plants Versus Animals

Plants, unlike animals, exhibit a very high degree of plasticity in their growth and development and employ diverse strategies to cope with the variations during diurnal cycles and stressful conditions. Plants and animals, despite their remarkable morphological and physiological differences, share many basic cellular processes and regulatory mechanisms. Alternative splicing (AS) is one such gene regulatory mechanism that modulates gene expression in multiple ways. It is now well established that AS is prevalent in all multicellular eukaryotes including plants and humans. Emerging evidence indicates that in plants, as in animals, transcription and splicing are coupled. Here, we reviewed recent evidence in support of co-transcriptional splicing in plants and highlighted similarities and differences between plants and humans. An unsettled question in the field of AS is the extent to which splice isoforms contribute to protein diversity. To take a critical look at this question, we presented a comprehensive summary of the current status of research in this area in both plants and humans, discussed limitations with the currently used approaches and suggested improvements to current methods and alternative approaches. We end with a discussion on the potential role of epigenetic modifications and chromatin state in splicing memory in plants primed with stresses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic ‘gatekeeper’ enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp–Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Å resolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg–Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

PTBP1 mRNA isoforms and regulation of their translation

Polypyrimidine tract-binding proteins (PTBPs) are RNA binding proteins that regulate a number of posttranscriptional events. Human PTBP1 transits between the nucleus and cytoplasm and is thought to regulate RNA processes in both. However, information about PTBP1 mRNA isoforms and regulation of PTPB1 expression remains incomplete. Here we mapped the major PTBP1 mRNA isoforms in HEK293T cells and identified alternative 5' and 3' untranslated regions (5'-UTRs, 3'-UTRs), as well as alternative splicing patterns in the protein coding region. We also assessed how the observed PTBP1 mRNA isoforms contribute to PTBP1 expression in different phases of the cell cycle. Previously, PTBP1 mRNAs were shown to crosslink to eukaryotic translation initiation factor 3 (eIF3). We find that eIF3 binds differently to each PTBP1 mRNA isoform in a cell cycle dependent manner. We also observe a strong correlation between eIF3 binding to PTBP1 mRNAs and repression of PTBP1 levels during the S phase of the cell cycle. Our results provide evidence of translational regulation of PTBP1 protein levels during the cell cycle, which may affect downstream regulation of alternative splicing and translation mediated by PTBP1 protein isoforms.

3′-UTR↗
DOE OSTI2024

A phosphorylation-deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana , rescuing mutant defects from global translation and gene expression to photosynthesis and growth

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from Arabidopsis thaliana, which indicate that plants expressing only a phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in both rps6a and rps6b genes when expressed at approximately twice the wild-type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl-terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho-enabled version of eS6z, the phospho-deficient seedlings retained a mild pointed-leaf phenotype, root growth was reduced, and certain cell cycle-related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects. There was little or no evidence for gain-of-function defects. As previously published, the phospho-deficient eS6z also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho-deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

60 APPLIED LIFE SCIENCES↗