Serine/arginine-rich Research Articles

Posttranslational splicing modifications as a key mechanism in cytarabine resistance in acute myeloid leukemia

Despite the approval of several drugs for AML, cytarabine is still widely used as a therapeutic approach. However, 85% of patients show resistance and only 10% overcome the disease. Using RNA-seq and phosphoproteomics, we show that RNA splicing and serine-arginine-rich (SR) proteins phosphorylation were altered during cytarabine resistance. Moreover, phosphorylation of SR proteins at diagnosis were significantly lower in responder than non-responder patients, pointing to their utility to predict response. These changes correlated with altered transcriptomic profiles of SR protein target genes. Notably, splicing inhibitors were therapeutically effective in treating sensitive and resistant AML cells as monotherapy or combination with other approved drugs. H3B-8800 and venetoclax combination showed the best efficacy in vitro, demonstrating synergistic effects in patient samples and no toxicity in healthy hematopoietic progenitors. Our results establish that RNA splicing inhibition, alone or combined with venetoclax, could be useful for the treatment of newly diagnosed or relapsed/refractory AML.

The functions of a 5' tRNA-Ala-derived fragment in gene expression.

Transfer RNA (tRNA) can produce smaller RNA fragments called tRNA-derived fragments (tRFs). tRFs play critical roles in multiple cellular programs, although the functional mechanisms of tRFs remain largely unknown in plants. In this study, we examined the phenotype associated with 5' tRF-Ala (tRF-Ala, produced from tRNA-Ala) overexpression and knockdown lines (tDR-Ala-OE and tDR-Ala-kd, respectively) and the mechanisms by which tRF-Ala affects mRNA levels in Arabidopsis (Arabidopsis thaliana). We investigated the candidate proteins associated with tRF-Ala by quantitative proteomics and confirmed the direct interaction between tRF-Ala and the splicing factor SERINE-ARGININE RICH PROTEIN 34 (SR34). A transcriptome sequencing analysis showed that 318 genes among all the genes (786) with substantial alternative splicing (AS) variance in tDR-Ala-OE lines are targets of SR34. tRF-Ala diminished the binding affinity between SR34 and its targets by direct competition for interaction with SR34. These findings reveal the critical roles of tRF-Ala in regulating mRNA levels and splicing.

The Splicing Factor OsSCL26 Regulates Phosphorus Homeostasis in Rice.

Phosphorus (P) is an essential nutrient for plant growth. However, its deficiency poses a significant challenge for crop production. To overcome the low P availability, plants have developed various strategies to regulate their P uptake and usage. In this study, we identified a splicing factor, OsSCL26, belonging to the Serine/arginine-rich (SR) proteins, that plays a crucial role in regulating P homeostasis in rice. OsSCL26 is expressed in the roots, leaves, and base nodes, with higher expression levels observed in the leaf blades during the vegetative growth stage. The OsSCL26 protein is localized in the nucleus. Mutation of OsSCL26 resulted in the accumulation of P in the shoot compared to the wild-type, and the dwarf phenotype of the osscl26 mutant was alleviated under low P conditions. Further analysis revealed that the accumulated P concentrations in the osscl26 mutant were higher in the old leaves and lower in the new leaves. Furthermore, the P-related genes, including the PHT and SPX family genes, were upregulated in the osscl26 mutant, and the exclusion/inclusion ratio of the two genes, OsSPX-MFS2 and OsNLA2, was increased compared to wild-type rice. These findings suggest that the splicing factor OsSCL26 plays a pivotal role in maintaining P homeostasis in rice by influencing the absorption and distribution of P through the regulation of the transcription and splicing of the P transport genes.

Repeat RNA structure and protein interactions influence RAN translation and neurodegenerative phenotypes in models of FTD and ALS

AbstractBackgroundThe most common genetic cause of Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) is an intronic GGGGCC hexanucleotide repeat expansion in C9orf72 (C9FTD/ALS). Transcribed GGGGCC repeat RNAs sequester RNA‐binding proteins (RBPs) into nuclear and cytoplasmic foci, which inhibits their normal functions. GGGGCC repeat RNAs also support translation of toxic repeat peptides through a process known as repeat associated non‐AUG (RAN) translation. Here we sought to understand how repeat RNA secondary structure and RBP engagements that occur during RNA transcription, processing, and trafficking influence GGGGCC RAN translation and repeat elicited toxicity, with a goal of identifying novel therapeutic targets.MethodWe developed a cellular RNA‐capture methodology coupled to mass spectrometry to identify GGGGCC repeat RNA‐interacting proteins within specific cellular compartments. In parallel, we used chemical approaches to probe GGGGCC repeat RNA secondary structures inside cells and RAN translation assays to determine whether repeat RNA secondary structures and/or interacting RBPs influence RAN translation efficiency. We employed Drosophila and neuronal models of repeat expansions to evaluate potential RBPs as disease modifiers.ResultOur RNA structural studies revealed that GGGGCC repeat RNAs form primarily hairpin structures, rather than G‐quadruplex structures, in cells. Folding repat RNAs into such hairpins supports RAN translation whereas folding repeat RNAs into g‐quadruplex structures strongly inhibits their translation. We identified multiple RBPs enriching in the nuclear fraction of GGGGCC RNA‐interactome, including hnRNPH1 and SR (serine/arginine‐rich domain) proteins, which are known components of the nuclear foci found in C9FTD/ALS patient cells. Our comparative analysis found that lowering SR protein expression or modifying its phosphorylation modified toxicity in Drosophila models of multiple repeat expansion disorders. Currently we are investigating how repeat RNA structures influence RBP interactions and whether structure dependent RBP interactions selectively modulate RAN translation.ConclusionWe present a comprehensive approach to evaluate the roles of repeat RNA secondary structures and critical RBPs in RNA toxicity underlying dementia‐associated repeat expansion disorders.

Plant serine/arginine-rich proteins: versatile players in RNA processing.

Serine/arginine-rich (SR) proteins participate in RNA processing by interacting with precursor mRNAs or other splicing factors to maintain plant growth and stress responses. Alternative splicing is an important mechanism involved in mRNA processing and regulation of gene expression at the posttranscriptional level, which is the main reason for the diversity of genes and proteins. The process of alternative splicing requires the participation of many specific splicing factors. The SR protein family is a splicing factor in eukaryotes. The vast majority of SR proteins' existence is an essential survival factor. Through its RS domain and other unique domains, SR proteins can interact with specific sequences of precursor mRNA or other splicing factors and cooperate to complete the correct selection of splicing sites or promote the formation of spliceosomes. They play essential roles in the composition and alternative splicing of precursor mRNAs, providing pivotal functions to maintain growth and stress responses in animals and plants. Although SR proteins have been identified in plants for three decades, their evolutionary trajectory, molecular function, and regulatory network remain largely unknown compared to their animal counterparts. This article reviews the current understanding of this gene family in eukaryotes and proposes potential key research priorities for future functional studies.

Cassava MeRS40 is required for the regulation of plant salt tolerance

Soil salinity affects the expression of serine/arginine-rich (SR) genes and isoforms by alternative splicing, which in turn regulates the adaptation of plants to stress. We previously identified the cassava spliceosomal component 35 like (SCL) and SR subfamilies, belonging to the SR protein family, which are extensively involved in responses to abiotic stresses. However, the post-transcriptional regulatory mechanism of cassava arginine/serine-rich (RS) subfamily in response to salt stress remains to be explored. In the current study, we identified 37 genes of the RS subfamily from 11 plant species and systematically investigated the transcript levels of the RS40 and RS31 genes under diverse abiotic stress conditions. Subsequently, an analysis of the conserved protein domains revealed that plant RS subfamily genes were likely to preserve their conserved molecular functions and played critical functional roles in responses to abiotic stresses. Importantly, we found that overexpression of MeRS40 in Arabidopsis enhanced salt tolerance by maintaining reactive oxygen species homeostasis and up-regulating the salt-responsive genes. However, overexpression of MeRS40 gene in cassava reduced salt tolerance due to the depression of its endogenous gene expression by negative autoregulation of its own pre-mRNA. Moreover, the MeRS40 protein interacted with MeU1-70Ks (MeU1-70Ka and MeU1-70Kb) in vivo and in vitro, respectively. Therefore, our ?ndings highlight the critical role of cassava SR proteins in responses to salt stress in plants.

Arabidopsis SRPKII family proteins regulate flowering via phosphorylation of SR proteins and effects on gene expression and alternative splicing.

Alternative splicing of pre-mRNAs is crucial for plant growth and development. Serine/arginine-rich (SR) proteins are a conserved family of RNA binding proteins that are critical for both constitutive and alternative splicing. However, how phosphorylation of SR proteins regulates gene transcription and alternative splicing during plant development is poorly understood. We found that the Arabidopsis thaliana L. SR protein-specific kinase II family proteins (SRPKIIs) play an important role in plant development, including flowering. SRPKIIs regulate the phosphorylation status of a subset of specific SR proteins, including SR45 and SC35, which subsequently mediates their subcellular localization. A phospho-dead SR45 mutant inhibits the assembly of the ASAP (apoptosis-and splicing-associated protein) complex and thereby upregulates the expression of FLC (FLOWERING LOCUS C) via epigenetic modification. The splicing efficiency of FLC introns was significantly increased in the shoot apex of the srpkii mutant. Transcriptomic analysis revealed that SRPKIIs regulate the alternative splicing of approximately four hundred genes, which largely overlap with those regulated by SR45 and SC35-SCL family proteins. In summary, we found that Arabidopsis SRPKIIs specifically affect the phosphorylation status of a subset SR proteins and regulate the expression and alternative splicing of FLC to control flowering time.

Investigation of the regulatory effects of synthesized antisense oligonucleotides on androgen receptor (AR) exon 3 splicing in prostate cancer cells

The Androgen Receptor (AR) gene plays a key role in castration-resistant prostate cancer (CRPC). Controlling the progression of CRPC by inhibiting AR gene expression is one of the core directions for prostate cancer (Pca) drug development. A 23-amino acids retention, named exon 3a, into the DNA binding domain of the splice variant AR23 has been shown to prevent AR from entering the nucleus and restore the sensitivity of cancer cells to related therapies. In this study, we conducted a preliminary investigation of the splicing modulation of the AR gene in order to develop a splice-switching therapy for Pca by promoting exon 3a inclusion. Using mutagenesis-coupled RT-PCR with AR minigene and over-expression of certain splicing factors, we found that serine/arginine-rich (SR) proteins are key factors facilitating the recognition of the 3′ splice site of exon 3a (L-3′ SS), while the deletion or blocking of the polypyrimidine tract (PPT) region of the original 3′ splice site of exon 3 (S-3′ SS) could strongly enhance exon 3a splicing without affecting the function of any SR protein. Furthermore, we designed a series of antisense oligonucleotides (ASOs) to screen drug candidates, and ASOs targeting S-3′ SS and its PPT region or the exonic region of exon 3 turned out to be most effective in rescuing exon 3a splicing. A dose–response test indicated ASO12 as the lead candidate drug significantly promoting the inclusion of exon 3a to more than 85%. MTT assay confirmed that the cell proliferation was significantly inhibited after ASO treatment. Our results provide the first glance to AR splicing regulation. With several promising therapeutic ASO candidates obtained here, further development of ASO drugs to treat CRPC is strongly encouraged.

Splicing factor-mediated regulation patterns reveals biological characteristics and aid in predicting prognosis in acute myeloid leukemia

BackgroundAlternative splicing (AS) of RNA is a fundamental biological process that shapes protein diversity. Many non-characteristic AS events are involved in the onset and development of acute myeloid leukemia (AML). Abnormal alterations in splicing factors (SFs), which regulate the onset of AS events, affect the process of splicing regulation. Hence, it is important to explore the relationship between SFs and the clinical features and biological processes of patients with AML.MethodsThis study focused on SFs of the classical heterogeneous nuclear ribonucleoprotein (hnRNP) family and arginine and serine/arginine-rich (SR) splicing factor family. We explored the relationship between the regulation patterns associated with the expression of SFs and clinicopathological factors and biological behaviors of AML based on a multi-omics approach. The biological functions of SRSF10 in AML were further analyzed using clinical samples and in vitro experiments.ResultsMost SFs were upregulated in AML samples and were associated with poor prognosis. The four splicing regulation patterns were characterized by differences in immune function, tumor mutation, signaling pathway activity, prognosis, and predicted response to chemotherapy and immunotherapy. A risk score model was constructed and validated as an independent prognostic factor for AML. Overall survival was significantly shorter in the high-risk score group. In addition, we confirmed that SRSF10 expression was significantly up-regulated in clinical samples of AML, and knockdown of SRSF10 inhibited the proliferation of AML cells and promoted apoptosis and G1 phase arrest during the cell cycle.ConclusionThe analysis of splicing regulation patterns can help us better understand the differences in the tumor microenvironment of patients with AML and guide clinical decision-making and prognosis prediction. SRSF10 can be a potential therapeutic target and biomarker for AML.

SGC-CLK-1: A chemical probe for the Cdc2-like kinases CLK1, CLK2, and CLK4

Small molecule modulators are important tools to study both basic biology and the complex signaling of protein kinases. The cdc2-like kinases (CLK) are a family of four kinases that have garnered recent interest for their involvement in a diverse set of diseases such as neurodegeneration, autoimmunity, and many cancers. Targeted medicinal chemistry around a CLK inhibitor hit identified through screening of a kinase inhibitor set against a large panel of kinases allowed us to identify a potent and selective inhibitor of CLK1, 2, and 4. Here, we present the synthesis, selectivity, and preliminary biological characterization of this compound – SGC-CLK-1 (CAF-170). We further show CLK2 has the highest binding affinity, and high CLK2 expression correlates with a lower IC50 in a screen of multiple cancer cell lines. Finally, we show that SGC-CLK-1 not only reduces serine arginine-rich (SR) protein phosphorylation but also alters SR protein and CLK2 subcellular localization in a reversible way. Therefore, we anticipate that this compound will be a valuable tool for increasing our understanding of CLKs and their targets, SR proteins, at the level of phosphorylation and subcellular localization.

Investigation of the effect of SRSF9 overexpression on HIV-1 production

Serine-arginine-rich splicing factors (SRSFs) are members of RNA processing proteins in the serine-arginine-rich (SR) family that could regulate the alternative splicing of the human immunodeficiency virus-1 (HIV-1). Whether SRSF9 has any effect on HIV-1 regulation requires elucidation. Here, we report for the first time the effects and mechanisms of SRSF9 on HIV-1 regulation. The overexpression of SRSF9 inhibits viral production and infectivity in both HEK293T and MT-4 cells. Deletion analysis of SRSF9 determined that the RNA regulation motif domain of SRSF9 is important for anti-HIV-1 effects. Furthermore, overexpression of SRSF9 increases multiple spliced forms of viral mRNA, such as Vpr mRNA. These data suggest that SRSF9 overexpression inhibits HIV-1 production by inducing the imbalanced HIV-1 mRNA splicing that could be exploited further for a novel HIV-1 therapeutic molecule. [BMB Reports 2022; 55(12): 639-644].

The rice blast fungus SR protein 1 regulates alternative splicing with unique mechanisms.

Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae. Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1, respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner.

ABA signaling prevents phosphodegradation of the SR45 splicing factor to alleviate inhibition of early seedling development in Arabidopsis

Serine/arginine-rich (SR) proteins are conserved splicing regulators that play important roles in plant stress responses, namely those mediated by the abscisic acid (ABA) hormone. The Arabidopsis thaliana SR-like protein SR45 is a described negative regulator of the ABA pathway during early seedling development. How the inhibition of growth by ABA signaling is counteracted to maintain plant development under stress conditions remains largely unknown. Here, we show that SR45 overexpression reduces Arabidopsis sensitivity to ABA during early seedling development. Biochemical and confocal microscopy analyses of transgenic plants expressing fluorescently tagged SR45 revealed that exposure to ABA dephosphorylates the protein at multiple amino acid residues and leads to its accumulation, due to SR45 stabilization via reduced ubiquitination and proteasomal degradation. Using phosphomutant and phosphomimetic transgenic Arabidopsis lines, we demonstrate the functional relevance of ABA-mediated dephosphorylation of a single SR45 residue, T264, in antagonizing SR45 ubiquitination and degradation to promote its function as a repressor of seedling ABA sensitivity. Our results reveal a mechanism that negatively autoregulates ABA signaling and allows early plant growth under stress via posttranslational control of the SR45 splicing factor.

Insights into sweet potato SR proteins: from evolution to species-specific expression and alternative splicing.

SR proteins from sweet potato have conserved functional domains and similar gene structures as that of Arabidopsis and rice in general. However, expression patterns and alternative splicing regulations of SR genes from different species have changed under stresses. Novel alternative splicing regulations were found in sweet potato SR genes. Serine/arginine-rich (SR) proteins play important roles in plant development and stress response by regulating the pre-mRNA splicing process. However, SR proteins have not been identified so far from an important crop sweet potato. Through bioinformatics analysis, our study identified 24 SR proteins from sweet potato, with comprehensively analyzing of protein characteristics, gene structure, chromosome localization, and cis-acting elements in promotors. Salt, heat, and mimic drought stresses triggered extensive but different expressional regulations on sweet potato SR genes. Interestingly, heat stress caused the most active disturbances in both gene transcription and pre-mRNA alternative splicing (AS). Tissue and species-specific transcriptional and pre-mRNA AS regulations in response to stresses were found in sweet potato, in comparison with Arabidopsis and rice. Moreover, novel patterns of pre-mRNA alternative splicing were found in SR proteins from sweet potato. Our study provided an insight into similarities and differences of SR proteins in different plant species from gene sequences to gene structures and stress responses, indicating SR proteins may regulate their downstream genes differently between different species and tissues by varied transcriptional and pre-mRNA AS regulations.

Regulatory Network of Serine/Arginine-Rich (SR) Proteins: The Molecular Mechanism and Physiological Function in Plants.

Serine/arginine-rich (SR) proteins are a type of splicing factor. They play significant roles in constitutive and alternative pre-mRNA splicing, and are involved in post-splicing activities, such as mRNA nuclear export, nonsense-mediated mRNA decay, mRNA translation, and miRNA biogenesis. In plants, SR proteins function under a complex regulatory network by protein–protein and RNA–protein interactions between SR proteins, other splicing factors, other proteins, or even RNAs. The regulatory networks of SR proteins are complex—they are regulated by the SR proteins themselves, they are phosphorylated and dephosphorylated through interactions with kinase, and they participate in signal transduction pathways, whereby signaling cascades can link the splicing machinery to the exterior environment. In a complex network, SR proteins are involved in plant growth and development, signal transduction, responses to abiotic and biotic stresses, and metabolism. Here, I review the current status of research on plant SR proteins, construct a model of SR proteins function, and ask many questions about SR proteins in plants.

Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly.

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.

Expansion and functional analysis of the SR-related protein family across the domains of life.

Serine/arginine-rich (SR) proteins comprise a family of proteins that is predominantly found in eukaryotes and plays a prominent role in RNA splicing. A characteristic feature of SR proteins is the presence of an S/R-rich low-complexity domain (RS domain), often in conjunction with spatially distinct RNA recognition motifs (RRMs). To date, 52 human proteins have been classified as SR or SR-related proteins. Here, using an unbiased series of composition criteria together with enrichment for known RNA binding activity, we identified >100 putative SR-related proteins in the human proteome. This method recovers known SR and SR-related proteins with high sensitivity (∼94%), yet identifies a number of additional proteins with many of the hallmark features of true SR-related proteins. Newly identified SR-related proteins display slightly different amino acid compositions yet similar levels of post-translational modification, suggesting that these new SR-related candidates are regulated in vivo and functionally important. Furthermore, candidate SR-related proteins with known RNA-binding activity (but not currently recognized as SR-related proteins) are nevertheless strongly associated with a variety of functions related to mRNA splicing and nuclear speckles. Finally, we applied our SR search method to all available reference proteomes, and provide maps of RS domains and Pfam annotations for all putative SR-related proteins as a resource. Together, these results expand the set of SR-related proteins in humans, and identify the most common functions associated with SR-related proteins across all domains of life.

The splicing factor 9G8 regulates the expression of NADPH-producing enzyme genes in Drosophila

Excess nutrients are stored as triglycerides, mostly as lipid droplets found in adipose tissue. Previous studies have characterized a group of splicing factors called serine/arginine rich (SR) proteins that function to identify intron/exon borders in regulating metabolic homeostasis in the Drosophila fat body. Decreasing the function of one SR protein, 9G8, causes an increase in triglyceride storage; however, the full complement of genes regulated by 9G8 to control metabolism is unknown. To address this question, we performed RNA sequencing on Drosophila fat bodies with 9G8 levels reduced by RNAi. Differential expression and differential exon usage analyses revealed several genes involved in the immune response, xenobiotic biology, protein translation, sleep, and lipid and carbohydrate metabolism whose expression or splicing is altered in 9G8-RNAi fat bodies. One gene that was both downregulated and had altered splicing in 9G8-RNAi fat bodies was Zwischenferment (Zw), the Drosophila homolog of human glucose 6-phosphate dehydrogenase (G6PD). G6PD regulates flux of glucose 6-phosphate (G6P) into the pentose phosphate pathway, which generates NADPH, a coenzyme for lipid synthesis. Interestingly, the other NADPH-producing enzyme genes in Drosophila (phosphogluconate dehydrogenase, isocitrate dehydrogenase and malic enzyme) were also decreased in 9G8-RNAi flies. Together, these findings suggest that 9G8 regulates several classes of genes and may regulate NADPH-producing enzyme genes to maintain metabolic homeostasis.

A Comprehensive Identification and Function Analysis of Serine/Arginine-Rich (SR) Proteins in Cotton (Gossypium spp.).

As one of the most important factors in alternative splicing (AS) events, serine/arginine-rich (SR) proteins not only participate in the growth and development of plants but also play pivotal roles in abiotic stresses. However, the research about SR proteins in cotton is still lacking. In this study, we performed an extensive comparative analysis of SR proteins and determined their phylogeny in the plant lineage. A total of 169 SR family members were identified from four Gossypium species, and these genes could be divided into eight distinct subfamilies. The domain, motif distribution and gene structure of cotton SR proteins are conserved within each subfamily. The expansion of SR genes is mainly contributed by WGD and allopolyploidization events in cotton. The selection pressure analysis showed that all the paralogous gene pairs were under purifying selection pressure. Many cis-elements responding to abiotic stress and phytohormones were identified in the upstream sequences of the GhSR genes. Expression profiling suggested that some GhSR genes may involve in the pathways of plant resistance to abiotic stresses. The WGCNA analysis showed that GhSCL-8 co-expressed with many abiotic responding related genes in a salt-responding network. The Y2H assays showed that GhSCL-8 could interact with GhSRs in other subfamilies. The subcellular location analysis showed that GhSCL-8 is expressed in the nucleus. The further VIGS assays showed that the silencing of GhSCL-8 could decrease salt tolerance in cotton. These results expand our knowledge of the evolution of the SR gene family in plants, and they will also contribute to the elucidation of the biological functions of SR genes in the future.

Altered metal distribution in the sr45-1 Arabidopsis mutant causes developmental defects.

The plant serine/arginine-rich (SR) splicing factor SR45 plays important roles in several biological processes, such as splicing, DNA methylation, innate immunity, glucose regulation, and abscisic acid signaling. A homozygous Arabidopsis sr45-1 null mutant is viable, but exhibits diverse phenotypic alterations, including delayed root development, late flowering, shorter siliques with fewer seeds, narrower leaves and petals, and unusual numbers of floral organs. Here, we report that the sr45-1 mutant presents an unexpected constitutive iron deficiency phenotype characterized by altered metal distribution in the plant. RNA-Sequencing highlighted severe perturbations in metal homeostasis, the phenylpropanoid pathway, oxidative stress responses, and reproductive development. Ionomic quantification and histochemical staining revealed strong iron accumulation in the sr45-1 root tissues accompanied by iron starvation in aerial parts. Mis-splicing of several key iron homeostasis genes, including BTS, bHLH104, PYE, FRD3, and ZIF1, was observed in sr45-1 roots. We showed that some sr45-1 developmental abnormalities can be complemented by exogenous iron supply. Our findings provide new insight into the molecular mechanisms governing the phenotypes of the sr45-1 mutant.