Splicing Targets Research Articles

Phase I First-in-Human Dose Escalation Study of the oral SF3B1 modulator H3B-8800 in myeloid neoplasms

We conducted a phase I clinical trial of H3B-8800, an oral small molecule that binds Splicing Factor 3B1 (SF3B1), in patients with MDS, CMML, or AML. Among 84 enrolled patients (42 MDS, 4 CMML and 38 AML), 62 were red blood cell (RBC) transfusion dependent at study entry. Dose escalation cohorts examined two once-daily dosing regimens: schedule I (5 days on/9 days off, range of doses studied 1–40 mg, n = 65) and schedule II (21 days on/7 days off, 7–20 mg, n = 19); 27 patients received treatment for ≥180 days. The most common treatment-related, treatment-emergent adverse events included diarrhea, nausea, fatigue, and vomiting. No complete or partial responses meeting IWG criteria were observed; however, RBC transfusion free intervals >56 days were observed in nine patients who were transfusion dependent at study entry (15%). Of 15 MDS patients with missense SF3B1 mutations, five experienced RBC transfusion independence (TI). Elevated pre-treatment expression of aberrant transcripts of Transmembrane Protein 14C (TMEM14C), an SF3B1 splicing target encoding a mitochondrial porphyrin transporter, was observed in MDS patients experiencing RBC TI. In summary, H3B-8800 treatment was associated with mostly low-grade TAEs and induced RBC TI in a biomarker-defined subset of MDS.

The Combined Human Genotype of Truncating TTN and RBM20 Mutations Is Associated with Severe and Early Onset of Dilated Cardiomyopathy.

A major cause of heart failure is cardiomyopathies, with dilated cardiomyopathy (DCM) as the most common form. Over 40 genes are linked to DCM, among them TTN and RBM20. Next Generation Sequencing in clinical DCM cohorts revealed truncating variants in TTN (TTNtv), accounting for up to 25% of familial DCM cases. Mutations in the cardiac splicing factor RNA binding motif protein 20 (RBM20) are also known to be associated with severe cardiomyopathies. TTN is one of the major RBM20 splicing targets. Most of the pathogenic RBM20 mutations are localized in the highly conserved arginine serine rich domain (RS), leading to a cytoplasmic mislocalization of mutant RBM20. Here, we present a patient with an early onset DCM carrying a combination of (likely) pathogenic TTN and RBM20 mutations. We show that the splicing of RBM20 target genes is affected in the mutation carrier. Furthermore, we reveal RBM20 haploinsufficiency presumably caused by the frameshift mutation in RBM20.

IRE1\u03b1 RIDD activity induced under ER stress drives neuronal death by the degradation of 14-3-3 \u03b8 mRNA in cortical neurons during glucose deprivation

Altered protein homeostasis is associated with neurodegenerative diseases and acute brain injury induced under energy depletion conditions such as ischemia. The accumulation of damaged or unfolded proteins triggers the unfolded protein response (UPR), which can act as a homeostatic response or lead to cell death. However, the factors involved in turning and adaptive response into a cell death mechanism are still not well understood. Several mechanisms leading to brain injury induced by severe hypoglycemia have been described but the contribution of the UPR has been poorly studied. Cell responses triggered during both the hypoglycemia and the glucose reinfusion periods can contribute to neuronal death. Therefore, we have investigated the activation dynamics of the PERK and the IRE1α branches of the UPR and their contribution to neuronal death in a model of glucose deprivation (GD) and glucose reintroduction (GR) in cortical neurons. Results show a rapid activation of the PERK/p-eIF2α/ATF4 pathway leading to protein synthesis inhibition during GD, which contributes to neuronal adaptation, however, sustained blockade of protein synthesis during GR promotes neuronal death. On the other hand, IRE1α activation occurs early during GD due to its interaction with BAK/BAX, while ASK1 is recruited to IRE1α activation complex during GR promoting the nuclear translocation of JNK and the upregulation of Chop. Most importantly, results show that IRE1α RNase activity towards its splicing target Xbp1 mRNA occurs late after GR, precluding a homeostatic role. Instead, IRE1α activity during GR drives neuronal death by positively regulating ASK1/JNK activity through the degradation of 14-3-3 θ mRNA, a negative regulator of ASK and an adaptor protein highly expressed in brain, implicated in neuroprotection. Collectively, results describe a novel regulatory mechanism of cell death in neurons, triggered by the downregulation of 14-3-3 θ mRNA induced by the IRE1α branch of the UPR.

Nanoindentation mechanical properties of CoCrFeNi high entropy alloy films

ABSTRACT CoCrFeNi high-entropy alloy films were deposited under different substrate bias by magnetron sputtering using a simple splicing target. The hardness, elastic modulus and creep properties of the films were measured by nanoindentation. The results showed that with the increase in substrate bias, CoCrFeNi films were always mainly composed of FCC phase and demonstrated smooth and dense surface with uniform distribution of elements, but the preferred orientation of the films changed from FCC (200) to no preferred and then to FCC (111). The hardness and elastic modulus of CoCrFeNi films were not sensitive to the strain rate of nanoindentation (0.05–0.15 s−1). The maximum hardness of the film was about 10.4 GPa under the substrate bias of –50 V, while the maximum elastic modulus of 249.8 GPa appeared under the substrate bias of –200 V. The applied negative substrate bias (−50 V ~ –200 V) could improve the elastic modulus and creep properties of the films.

Two Isoforms of Eya3 Influence Muscle Cell Differentiation and Become Dysregulated in Striated Muscle Diseases

Alternative splicing is a ubiquitous gene regulatory mechanism by which diverse transcripts are derived from a finite genome. During development, complex splicing transitions occur, leading to varied transcript profiles in fetal and adult tissues. Like gene expression, splicing often becomes dysregulated in cancer, neurological, and muscular diseases. One barrier to therapeutic targeting of alternative splicing is that the function of splice isoforms, both physiologically and pathologically, are vastly understudied. Here, we begin to address this knowledge gap by describing an evolutionarily conserved alternative splicing event in eyes absent homolog 3 (Eya3) gene for the first time. The short isoform of Eya3 is a dual threonine- and tyrosine-phosphatase that is known to promote cell growth and chemotherapeutic resistance. By analyzing RNA-sequencing data from various mouse tissues, we identified the long isoform of Eya3, which contains an additional 138nt, representing a 46 amino acid in-frame insertion immediately following the threonine phosphatase domain. Using reverse transcription-PCR (RT-PCR) of mouse, human, and rabbit tissues, we discovered that Eya3 splicing is tissue-specific, with the long isoform expressed exclusively in the heart, skeletal muscle, and brain. Additionally, we found that during heart and skeletal muscle development, expression of the long Eya3 isoform gradually increases relative to the expression of the short isoform. In myotonic dystrophy type 1 patient hearts, and failing mouse hearts, the short Eya3 isoform is re-expressed, illustrating that Eya3 splicing is dysregulated in striated muscle diseases. To understand Eya3 splicing in further molecular detail, we utilized the well-established C2C12 murine myoblast cell line. Differentiation of myoblasts to myotubes robustly recapitulates the Eya3 splicing transition observed during tissue development, making them an appropriate cell culture system for our work. Depletion of Eya3 via siRNA transfection inhibited myoblast proliferation and fusion. Interestingly, blocking the endogenous Eya3 alternative splicing transition with morpholino antisense oligonucleotides also suppressed normal differentiation as determined by immunohistochemical staining and confocal microscopy. These results indicate that Eya3, and its splice isoforms, play an important role in early muscle development. In summary, our studies uncover a developmentally-regulated, tissue-specific, and disease-relevant splicing event in Eya3. Moving forward, we seek to determine the molecular mechanism by which Eya3 isoforms regulate normal muscle development and to evaluate Eya3 as a potential therapeutic target.

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing.

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3’ UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons.

Abstract PD15-07: Myc regulates alternative splicing through a network of RNA binding proteins in breast cancer

Abstract The oncogenic transcription factor MYC is one of the most frequently altered genes in cancer and is highly dysregulated in aggressive tumor types, including triple negative breast cancer (TNBC). Although direct targeting of MYC or its regulators in human tumors has proved difficult, the undisputed clinical relevance of this transcription factor warrants continued study for therapeutic targeting of MYC-driven processes in tumors. Recently, we and others have shown the tumorigenic capacity of MYC is critically dependent on the RNA splicing machinery and on several splicing factor RNA-binding proteins (RBPs). Dysregulation of alternative mRNA splicing, a key step in gene expression regulation, is a hallmark of cancer, and can lead to the expression of isoforms driving tumor initiation, progression or drug resistance. Understanding which component(s) of the RNA machinery are regulated by MYC, and the functions of MYC-induced spliced isoforms, may reveal new targets that circumvent prior failures of directly targeting MYC. Here, we examine MYC-induced changes in gene expression and alternative splicing through RNA-sequencing of breast cell lines and tumors. Using a mammary epithelial cell line with an inducible MYC system, we identified over 4,000 MYC-induced alternative splicing events as well as 378 MYC-induced RBPs. Co-expression network analysis of these RBPs in TCGA breast tumor RNA-seq data uncovered 8 RBP modules, four of which are highly correlated with MYC activity and highly expressed in TNBC. One of these modules is preserved across almost all other TCGA tumor types and remains highly correlated with MYC activity, suggesting a pan-cancer role of this co-expression module. We are now defining the functional role of these RBP modules in MYC-driven tumorigenesis in breast cancer models, as well as identifying their downstream oncogenic splicing targets. Our research project will define the role and regulation of alternative splicing in breast cancer, and more specifically in aggressive tumor types such as TNBC. Our findings will provide mechanistic insight into the role of MYC-driven splicing in tumor maintenance and will uncover novel therapeutic targets for MYC-active tumors. Citation Format: Laura Urbanski, Brittany Angarola, Mattia Brugiolo, Marina Yurieva, Sunghee Park, Olga Anczukow. Myc regulates alternative splicing through a network of RNA binding proteins in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-07.

Rbm24 regulates inner-ear-specific alternative splicing and is essential for maintaining auditory and motor coordination

ABSTRACT Tissue-specific alternative splicing (AS) is emerging as one of the most exciting types of mechanisms associated with organ development and disease. In the auditory system, many hearing-related genes undergo AS, and errors in this process result in syndromic or non-syndromic hearing loss. However, little is known about the factors and mechanisms directing AS in the inner ear. In the present study, we identified a novel RNA-binding protein, Rbm24, which was critically involved in regulating inner-ear-specific AS. Rbm24 deletion resulted in hearing loss and defects in motor coordination. Global splicing analysis showed Rbm24 was required for correct splicing of a subset of pre-mRNA transcripts with essential roles in stereocilia integrity and survival of hair cells. Furthermore, we identified that Rbm24 directly regulated the splicing of Cdh23, a known disease gene responsible for human Usher syndrome 1D and non-syndromic autosomal recessive deafness DFNB12. In conclusion, our findings demonstrated that Rbm24 was a critical factor in regulating inner-ear-specific splicing and maintaining the hearing and motor coordination function of the inner ear. Our data not only offer mechanistic insights but also provide functional annotation of Rbm24 splicing targets that contribute to hearing loss.

Abstract 3747: Dysregulated splicing of Annexin A7 impairs receptor tyrosine kinase trafficking in glioblastoma

Abstract Alternative splicing (AS), a tightly regulated process, contributes to proteome diversification essential for tissue development and identity. During brain development, splicing targets include genes involved in several cellular processes like endocytosis, cytoskeleton dynamics and vesicle-mediated transport. Deregulation of AS in cancers enables tumor cells to alter the transcriptome in favor of isoforms that drive tumor progression. Annexin A7 (ANXA7) is a hydrophilic protein that binds membranes in a calcium-dependent manner; it is important for membrane scaffolding and vesicle trafficking. ANXA7 is alternatively spliced by PTBP1 and expressed as two isoforms - unspliced ANXA7 Isoform 1 (I1), containing cassette exon 6 and spliced Isoform 2 (I2). We determined that this splicing of ANXA7 is lineage-specific in the brain - I1 is highly expressed in post-mitotic mature neurons while I2 is expressed in neural and glial precursors. In order to understand the functional impact of these isoforms, we generated GBM cell lines that express endogenous I2 (I2 cells), or endogenous I2 + exogenous I1 (I1 cells). We evaluated the influence of I1 and/or I2 expression on RTK levels, activation and downstream events using western blot and co-immunoprecipitation assays. Receptor trafficking was observed using flow cytometry and immunofluorescence assays tagging different components of the endocytic pathway. In GBM, we observed high levels of PTBP1 with a subsequent increase in I2 levels indicative of dysregulated AS. Consequently, we observed upregulated activation of several receptor tyrosine kinases (RTKs) such as EGFR, MET, PDGFRα and EGFR vIII in the I2 cells. In I1 cells, however, we observed reduced levels and activation of all aforementioned RTKs along with the diminished activation of downstream pathways. Using EGFR signaling as a model, we found that in I1 cells EGFR co-localized with markers of lysosomal degradation indicating efficient trafficking of activated EGFR for endosomal degradation; co-localization was absent in I2 cells indicative of impaired trafficking. We propose that I1, by virtue of the amino acids encoded by cassette exon 6, binds to conserved motifs within multiple RTKs and targets them for endosomal degradation thereby attenuating tumorigenic signaling in GBM. Our results show that ANXA7 I1 is tumor suppressive in GBM and that loss of I1 promotes tumorigenicity by perpetuating RTK signaling. We present a mechanism that explains, in part, how I1 mediates the degradation of multiple tumorigenic RTKs by spatio-temporally regulating their endosomal trafficking. A refined understanding of how I1 functions will provide the foundation for future, selective therapies that target dysregulated AS in GBM. Citation Format: Sindhu Nair, Rajani Rajbhandari, Centdrika Dates-Hurt, Markus Bredel. Dysregulated splicing of Annexin A7 impairs receptor tyrosine kinase trafficking in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3747.

RNA Binding Motif 5 (RBM5) in the CNS-Moving Beyond Cancer to Harness RNA Splicing to Mitigate the Consequences of Brain Injury.

Gene splicing modulates the potency of cell death effectors, alters neuropathological disease processes, influences neuronal recovery, but may also direct distinct mechanisms of secondary brain injury. Therapeutic targeting of RNA splicing is a promising avenue for next-generation CNS treatments. RNA-binding proteins (RBPs) regulate a variety of RNA species and are prime candidates in the hunt for druggable targets to manipulate and tailor gene-splicing responses in the brain. RBPs preferentially recognize unique consensus sequences in targeted mRNAs. Also, RBPs often contain multiple RNA-binding domains (RBDs)—each having a unique consensus sequence—suggesting the possibility that drugs could be developed to block individual functional domains, increasing the precision of RBP-targeting therapies. Empirical characterization of most RBPs is lacking and represents a major barrier to advance this emerging therapeutic area. There is a paucity of data on the role of RBPs in the brain including, identification of their unique mRNA targets, defining how CNS insults affect their levels and elucidating which RBPs (and individual domains within) to target to improve neurological outcomes. This review focuses on the state-of-the-art of the RBP tumor suppressor RNA binding motif 5 (RBM5) in the CNS. We discuss its potent pro-death roles in cancer, which motivated our interest to study it in the brain. We review recent studies showing that RBM5 levels are increased after CNS trauma and that it promotes neuronal death in vitro. Finally, we conclude with recent reports on the first set of RBM5 regulated genes identified in the intact brain, and discuss how those findings provide new clues germane to its potential function(s) in the CNS, and pose new questions on its therapeutic utility to mitigate CNS injury.

Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.

RALF1-FERONIA complex affects splicing dynamics to modulate stress responses and growth in plants.

The environmentally responsive signaling pathways that link global transcriptomic changes through alternative splicing (AS) to plant fitness remain unclear. Here, we found that the interaction of the extracellular rapid alkalinization FACTOR 1 (RALF1) peptide with its receptor FERONIA (FER) triggered a rapid and massive RNA AS response by interacting with and phosphorylating glycine-rich RNA binding protein7 (GRP7) to elevate GRP7 nuclear accumulation in Arabidopsis thaliana. FER-dependent GRP7 phosphorylation enhanced its mRNA binding ability and its association with the spliceosome component U1-70K to enable splice site selection, modulating dynamic AS. Genetic reversal of a RALF1-FER-dependent splicing target partly rescued mutants deficient in GRP7. AS of GRP7 itself induced nonsense-mediated decay feedback to the RALF1-FER-GRP7 module, fine-tuning stress responses, and cell growth. The RALF1-FER-GRP7 module provides a paradigm for regulatory mechanisms of RNA splicing in response to external stimuli.

Modulator-Dependent RBPs Changes Alternative Splicing Outcomes in Kidney Cancer.

Alternative splicing alterations can contribute to human disease. The ability of an RNA-binding protein to regulate alternative splicing outcomes can be modulated by a variety of genetic and epigenetic mechanisms. In this study, we use a computational framework to investigate the roles of certain genes, termed modulators, on changing RBPs’ effect on splicing regulation. A total of 1,040,254 modulator-mediated RBP-splicing interactions were identified, including 137 RBPs, 4,309 splicing events and 2,905 modulator candidates from TCGA-KIRC RNA sequencing data. Modulators function categories were defined according to the correlation changes between RBPs expression and their targets splicing outcomes. QKI, as one of the RBPs influencing the most splicing events, attracted our attention in this study: 2,014 changing triplets were identified, including 1,101 modulators and 187 splicing events. Pathway enrichment analysis showed that QKI splicing targets were enriched in tight junction pathway, endocytosis and MAPK signaling pathways, all of which are highly associated with cancer development and progression. This is the first instance of a comprehensive study on how alternative splicing outcomes changes are associated with different expression level of certain proteins, even though they were regulated by the same RBP. Our work may provide a novel view on understanding alternative splicing mechanisms in kidney cancer.

HNRNPA1-induced spliceopathy in a transgenic mouse model of myotonic dystrophy

Studies on myotonic dystrophy type 1 (DM1) have led to the RNA-mediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUGexp) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse HSALR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUGexp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.

Effects of nitriding on the microstructure and properties of VAlTiCrMo high-entropy alloy coatings by sputtering technique

The VAlTiCrMo and (VAlTiCrMo)Nx coatings were prepared by sputtering V–Al–Ti–Cr–Mo splicing targets. The microstructure and chemical composition were analyzed by means of X-ray diffraction (XRD), scanning electron microscope (SEM), energy dispersive spectrometer (EDS) and transmission electron microscope (TEM). The high-entropy alloy coating and the corresponding nitride coating have body-centered cubic (BCC) and face-centered cubic (FCC) structures respectively. Nitriding changes the phase structure and growth mode of the high-entropy coating, and the synthesized high-entropy nitride coatings are more compact. Meanwhile, the introduction of nitrogen leads to severe lattice distortion, which improves the mechanical properties of the high-entropy coating. As the nitriding degree increases, the corrosion resistance of the high-entropy nitride coatings in 3.5 wt% NaCl solution is improved. The synthesized (VAlTiCrMo)Nx coating exhibits optimized mechanical properties and nobler corrosion resistance when the N2 flow is 800 sccm.

The RNA-binding protein AKAP8 suppresses tumor metastasis by antagonizing EMT-associated alternative splicing

Alternative splicing has been shown to causally contribute to the epithelial–mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein–protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.

Proces składania RNA jako cel oddziaływania terapeutycznego

Proces składania RNA jako cel oddziaływania terapeutycznego

BLISTER-regulated vegetative growth is dependent on the protein kinase domain of ER stress modulator IRE1A in Arabidopsis thaliana.

The unfolded protein response (UPR) is required for protein homeostasis in the endoplasmic reticulum (ER) when plants are challenged by adverse environmental conditions. Inositol-requiring enzyme 1 (IRE1), the bifunctional protein kinase / ribonuclease, is an important UPR regulator in plants mediating cytoplasmic splicing of the mRNA encoding the transcription factor bZIP60. This activates the UPR signaling pathway and regulates canonical UPR genes. However, how the protein activity of IRE1 is controlled during plant growth and development is largely unknown. In the present study, we demonstrate that the nuclear and Golgi-localized protein BLISTER (BLI) negatively controls the activity of IRE1A/IRE1B under normal growth condition in Arabidopsis. Loss-of-function mutation of BLI results in chronic up-regulation of a set of both canonical UPR genes and non-canonical UPR downstream genes, leading to cell death and growth retardation. Genetic analysis indicates that BLI-regulated vegetative growth phenotype is dependent on IRE1A/IRE1B but not their canonical splicing target bZIP60. Genetic complementation with mutation analysis suggests that the D570/K572 residues in the ATP-binding pocket and N780 residue in the RNase domain of IRE1A are required for the activation of canonical UPR gene expression, in contrast, the D570/K572 residues and D590 residue in the protein kinase domain of IRE1A are important for the induction of non-canonical UPR downstream genes in the BLI mutant background, which correlates with the shoot growth phenotype. Hence, our results reveal the important role of IRE1A in plant growth and development, and BLI negatively controls IRE1A’s function under normal growth condition in plants.

Alternative splicing in aging and longevity.

Alternative pre-mRNA splicing increases the complexity of the proteome that can be generated from the available genomic coding sequences. Dysregulation of the splicing process has been implicated in a vast repertoire of diseases. However, splicing has recently been linked to both the aging process itself and pro-longevity interventions. This review focuses on recent research towards defining RNA splicing as a new hallmark of aging. We highlight dysfunctional alternative splicing events that contribute to the aging phenotype across multiple species, along with recent efforts toward deciphering mechanistic roles for RNA splicing in the regulation of aging and longevity. Further, we discuss recent research demonstrating a direct requirement for specific splicing factors in pro-longevity interventions, and specifically how nutrient signaling pathways interface to splicing factor regulation and downstream splicing targets. Finally, we review the emerging potential of using splicing profiles as a predictor of biological age and life expectancy. Understanding the role of RNA splicing components and downstream targets altered in aging may provide opportunities to develop therapeutics and ultimately extend healthy lifespan in humans.

Corrosion behavior of diamond-like carbon film induced by Al/Ti co-doping

Diamond-like carbon films with Al and Ti dopants (Al/Ti-DLC) were fabricated by a hybrid ion beam deposition system combined with a special design of splicing targets, in which the Al/Ti ratio was changed from 4.0 to 1.1 by tailoring the Al content. The structural evolution and corrosion behavior of Al/Ti-DLC films in 3.5 wt% NaCl solution were systematically investigated by comparing with the 316L SS and pure DLC film. Results revealed that compared with the pure DLC and 316L SS substrate, the Al/Ti-DLC film exhibited enhanced corrosion resistance by reducing the porosity and increasing the barrier for both the charge transfer and the diffusion of corrosive ions into the substrate. In particular, the anti-corrosion behavior of DLC film highly depended on the Al/Ti ratio, which decreased gradually with the Al/Ti ratio ranged from 4.0 to 1.1. Structural and EIS analysis indicated that this was ascribed to both the increased residual stress and sp2 hybridized fraction, which induced not only the formation and propagation of cracks during the corrosion process but also the high electric conductivity and low diffusion barrier of corrosive ions, accelerating the corrosion processes at the film/substrate interface.