Variation of Macro- and Microelements, and Trace Metals in Spring Wheat Genetic Resources in Siberia

Western Siberia is one of the major spring wheat regions of Russia, cultivating over 7 Mha. The objective of the study was to evaluate the variation of macro- and microelements, and of trace metals in four distinct groups of genetic resources: primary synthetics from CIMMYT (37 entries), primary synthetics from Japan (8), US hard red spring wheat cultivars (14), and material from the Kazakhstan–Siberian Network on Spring Wheat Improvement (KASIB) (74). The experiment was conducted at Omsk State Agrarian University, using a random complete block design with four replicates in 2017 and 2018. Concentrations of 15 elements were included in the analysis: macroelements, Ca, K, Mg, P, and S; microelements, Fe, Cu, Mn, and Zn; toxic trace elements, Cd, Co, Ni; and trace elements, Mo, Rb, and Sr. Protein content was found to be positively correlated with the concentrations of 11 of the elements in one or both years. Multiple regression was used to adjust the concentration of each element, based on significant correlations with agronomic traits and macroelements. All 15 elements were evaluated for their suitability for genetic enhancement, considering phenotypic variation, their share of the genetic component in this variation, as well as the dependence of the element concentration on other traits. Three trace elements (Sr, Mo, and Co) were identified as traits that were relatively easy to enhance through breeding. These were followed by Ca, Cd, Rb, and K. The important biofortification elements Mn and Zn were among the traits that were difficult to enhance genetically. The CIMMYT and Japanese synthetics had significantly higher concentrations of K and Sr, compared to the local check. The Japanese synthetics also had the highest concentrations of Ca, S, Cd, and Mo. The US cultivars had concentrations of Ca as high as the Japanese synthetics, and the highest concentrations of Mg and Fe. KASIB’s germplasm had near-average values for most elements. Superior germplasm, with high macro- and microelement concentrations and low trace-element concentrations, was found in all groups of material included.

Mechanisms of modulation of pre-mRNA 3D structural scaffolds for splicing substrate definition

Coordination of different serine-arginine-rich (SR) proteins - a class of critical splicing activators - facilitates recognition of the highly degenerate cognate splice signal sequences against the background sequences. Yet, the mechanistic details of their actions remain unclear. Here we show that cooperative binding of SR proteins to exonic and intronic motifs remodels the pre-mRNA 3D structural scaffold. The scaffold generated by pre-mRNA-specific combinations of different SR proteins in an appropriate stoichiometry is recognized by U1 snRNP. A large excess of U1 snRNP particles displaces the majority of the bound SR protein molecules from the remodeled pre-mRNA. A higher than optimal stoichiometry of SR proteins occludes the binding sites on the pre-mRNA, raising the U1 snRNP levels required for SR protein displacement and potentially impeding spliceosome assembly. This novel step is important for distinguishing the substrate and the non-substrate by U2AF65 - the primary 3' splice site-recognizing factor. Overall, this work elucidates early regulatory steps of mammalian splicing substrate definition by SR proteins.

Modulation of RNA Splicing Enhances Response to BCL2 Inhibition in Acute Myeloid Leukemia

Resistance to therapy is one of the most significant challenges in the treatment of acute myeloid leukemia (AML). While great efforts have uncovered genetic mechanisms of resistance to certain AML-directed therapies, to date, treatment resistance in AML has only partially explained by acquired genetic alterations. Here, we performed genome-wide CRISPR/Cas9 screens to identify drug-gene interactions that modulate therapeutic response to treatments commonly used in AML. Interestingly, our findings uncovered several genes that regulate pre-mRNA splicing whose loss strongly synergized with venetoclax, a BH3 mimetic that blocks the antiapoptotic protein BCL-2. To further delineate the role of RNA processing in response to AML treatments, we performed secondary CRISPR screens with a domain-focused gRNA library targeting 490 RNA processing factors in the presence of various AML drugs. Overall, these genetic screens identified a number of RNA splicing factors whose loss-of-function sensitized AML cells to BCL2 inhibition (Fig. A). Among the top gene candidates whose loss promoted venetoclax efficacy was the splicing factor RBM10 (Fig.B). Strikingly, loss of RBM10 exclusively synergized with venetoclax-based treatments across AML therapeutics, including in TP53 mutant lines (Fig.C-D). Moreover, RBM10 loss restored venetoclax sensitivity to AML cell line variants with acquired venetoclax resistance. Interestingly, while many RNA splicing factors are pan-essential, generation of an Rbm10 conditional knockout mouse revealed that Rbm10 is completely dispensable for steady-state normal hematopoiesis (Fig.E). Since RBM10 has not been studied previously in hematopoiesis, we mapped the impact of RBM10 on mRNA expression and splicing using RNA-seq and direct RNA binding partners genome-wide by eCLIP-Seq (Fig. F). RBM10 loss was strongly associated with downregulation of BCL2A1, an anti-apoptotic factor whose expression is correlated with venetoclax resistance in AML (Fig.G-H). This was dependent on RBM10's ability to bind RNA and expression of BCL2A1 cDNA fully rescued the growth-inhibitory effect of RBM10 KO-venetoclax treated AML cells. Overall, the above data support RBM10 as a synthetic lethal vulnerability in venetoclax therapy. Beyond RBM10, our genetic screens also identified several splicing factors belonging to the family of serine and arginine-rich (SR) proteins whose loss synergized with venetoclax treatment (Fig. I). SR proteins are essential for pre-mRNA splicing and are substrates for phosphorylation by conserved family of kinases, such as Cdc2-like kinases (CLKs) and (dual-specificity tyrosine-regulated kinases) DYRKs. We therefore utilized a series of selective pan-CLK/DYRK1A inhibitors, including SM09419 and SM08502, that potently suppress SR protein phosphorylation. Interestingly, BCL2 is one of the top genetic dependencies upon DYRK1A genetic suppression in prior work from the DepMap (Fig. J). Pharmacologic inhibition of CLK/DYRK1A exhibited high in vitro efficacy at nanomolar range across a diverse range of AML subtypes including cell lines with acquired venetoclax resistance (Fig.K). Consistent with this, combined SM09419 and venetoclax displayed synergistic anti-leukemic effects and venetoclax-sensitive AML cell lines (Fig.L). Taken together, these data support the notion of targeting CLK/DYRK1A in the context of BCL2 inhibition. In this study, we systematically defined gene interactions that mediate the response to a wide range of AML drugs. Recent studies have begun to show that dysfunctional RNA processing promotes AML development. However, the role of RNA processing in modulating drug responsiveness in AML is not well understood. Here, we have uncovered that synthetic lethal targeting of splicing factors, such as RBM10, increases sensitivity of AML cells to BCL2 inhibition. Therapeutically, pharmacologic inhibition of SR protein function via inhibiting CLK/DYRK1A-mediated phosphorylation of splicing factors is an effective strategy used in combination with venetoclax or to overcome venetoclax resistance. Overall, our findings underscore the central importance of RNA splicing in drug response and provides a therapeutic rationale for modulating RNA splicing to enhance current AML therapies. Figure 1 Figure 1. Disclosures McMillan: Prizer: Ended employment in the past 24 months. Bossard: Biosplice Therapeutics: Current Employment. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

The plant-specific SCL30a SR protein regulates ABA-dependent seed traits and salt stress tolerance during germination

SR (serine/arginine-rich) proteins are conserved RNA-binding proteins best known as key regulators of splicing, which have also been implicated in other steps of gene expression. Despite mounting evidence for their role in plant development and stress responses, the molecular pathways underlying SR protein regulation of these processes remain elusive. Here we show that the plant-specific SCL30a SR protein negatively regulates abscisic acid (ABA) signaling to control important seed traits and salt stress responses during germination in Arabidopsis. The SCL30a gene is upregulated during seed imbibition and germination, and its loss of function results in smaller seeds displaying enhanced dormancy and elevated expression of ABA-responsive genes as well as of genes repressed during the germination process. Moreover, the knockout mutant is hypersensitive to ABA and high salinity, while transgenic plants overexpressing SCL30a exhibit reduced ABA sensitivity and enhanced tolerance to salt stress during seed germination. An ABA biosynthesis inhibitor rescues the mutant's enhanced sensitivity to stress, and epistatic analyses confirm that this hypersensitivity requires a functional ABA pathway. Finally, seed ABA levels are unchanged by altered SCL30a expression, indicating that the SR protein positively regulates stress tolerance during seed germination by reducing sensitivity to the phytohormone. Our results reveal a new key player in ABA-mediated control of early development and stress response, and underscore the role of plant SR proteins as important regulators of the ABA signaling pathway.

FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1-dependent axis that controls VEGFR1 splicing in endothelial cells

Background Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. Results By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. Conclusions We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression.

An SR protein is essential for activating DNA repair in malaria parasites

Plasmodium falciparum, the parasite responsible for the deadliest form of human malaria, replicates within the erythrocytes of its host where it encounters numerous pressures that cause extensive DNA damage, which must be repaired efficiently to ensure parasite survival. Malaria parasites, which lost the NHEJ pathway for repairing DNA double strand breaks, have evolved unique mechanisms that enable them to robustly maintain genome integrity under such harsh conditions. However, the nature of these adaptations is unknown. We show that a highly conserved RNA splicing factor, PfSR1, plays an unexpected and crucial role in DNA repair in malaria parasites. Using an inducible and reversible system to manipulate PfSR1 expression, we demonstrate that this protein is recruited to foci of DNA damage. While loss of PfSR1 does not impair parasite viability, the protein is essential for parasites recovery from DNA damaging agents or exposure to artemisinin, the first line antimalarial drug, demonstrating its necessity for DNA repair. These findings provide key insights into the evolution of DNA repair pathways in malaria parasites as well as the parasite's ability to recover from antimalarial treatment.

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Post-translational modifications (PTMs) within splicing factor RNA-binding proteins (RBPs), such as phosphorylation, regulate several critical steps in RNA metabolism including spliceosome assembly, alternative splicing and mRNA export. Notably, the arginine-/serine-rich (RS) domains in SR proteins are densely modified by phosphorylation compared with the remainder of the proteome. Previously, we showed that dephosphorylation of SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich to the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. Phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high molecular weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine-/arginine protein kinase 2 (SRPK2) in vitro prevented high molecular weight SRSF2 species formation. Furthermore, we pharmacologically inhibited SRPKs in mammalian cells and observed increased cytoplasmic granules as well as the formation of cytoplasmic SRSF2 tubular structures that associate with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.

miR-150 and SRPK1 regulate AKT3 expression to participate in LPS-induced inflammatory response

miR-150 was found to target the 3′-untranslated regions of AKT3, and the AKT pathway was affected by SR protein kinase 1 (SRPK1). However, the expression and significance of miR-150, AKT3 and SRPK1 in acute lung injury (ALI) were not clear. Here, we found that the expression of miR-150 was significantly reduced, while the expression of AKT3 and SRPK1 were markedly increased in LPS-treated A549, THP-1 and RAW 264.7 cells. miR-150 significantly decreased levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, reduced the expression of AKT3, but had no impact on SRPK1 expression compared with the control group in LPS-treated A549, THP-1 and RAW 264.7 cells. AKT3 silencing only reduced the production of pro-inflammatory cytokines and showed no effect on miR-150 and SRPK1 expression. Finally, we observed that miR-150 mimics and/or silencing of SRPK1 decreased the expression of AKT3 mRNA. Besides, over-expression of miR-150 or silencing of SRPK1 also reduced the expression of AKT3 protein, which exhibited the lowest level in the miR-150 mimics plus si-SRPK1 group. However, si-SRPK1 had no effect on miR-150 level. In conclusion, miR-150 and SRPK1 separately and cooperatively participate into inflammatory responses in ALI through regulating AKT3 pathway. Increased miR-150 and silenced SRPK1 may be a novel potential factor for preventing and treating more inflammatory lung diseases.

CLK1 reorganizes the splicing factor U1-70K for early spliceosomal protein assembly

Early spliceosome assembly requires phosphorylation of U1-70K, a constituent of the U1 small nuclear ribonucleoprotein (snRNP), but it is unclear which sites are phosphorylated, and by what enzyme, and how such modification regulates function. By profiling the proteome, we found that the Cdc2-like kinase 1 (CLK1) phosphorylates Ser-226 in the C terminus of U1-70K. This releases U1-70K from subnuclear granules facilitating interaction with U1 snRNP and the serine-arginine (SR) protein SRSF1, critical steps in establishing the 5′ splice site. CLK1 breaks contacts between the C terminus and the RNA recognition motif (RRM) in U1-70K releasing the RRM to bind SRSF1. This reorganization also permits stable interactions between U1-70K and several proteins associated with U1 snRNP. Nuclear induction of the SR protein kinase 1 (SRPK1) facilitates CLK1 dissociation from U1-70K, recycling the kinase for catalysis. These studies demonstrate that CLK1 plays a vital, signal-dependent role in early spliceosomal protein assembly by contouring U1-70K for protein–protein multitasking.