Spliceosomal Proteins Research Articles

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3′ untranslated region (3′UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3′UTR to non-productive isoforms with a long 3′UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5′ splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3′UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.

Transcriptomic and proteomic insight into the effects of a defined European mistletoe extract in Ewing sarcoma cells reveals cellular stress responses

BackgroundThe hydrophobic triterpenes, oleanolic and betulinic acid as well as the hydrophilic mistletoe lectins and viscotoxins possess anticancer properties. They do all occur in combination in European mistletoe (Viscum album L.). Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We have previously shown that mistletoe lectins and triterpene acids are effective against Ewing sarcoma in vitro, ex vivo and in vivo.MethodsWe recreated a total mistletoe effect (viscumTT) by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilised with cyclodextrins and analysed the effects of viscumTT and the single extracts on TC-71 Ewing sarcoma cells in vitro by transcriptomic and proteomic profiling.ResultsTreatment with the extracts strongly impacted Ewing sarcoma cell gene and protein expression. Apoptosis-associated and stress-activated genes were upregulated, proteasomal protein abundance enhanced and ribosomal and spliceosomal proteins downregulated. The mechanism of action of viscum, TT and viscumTT in TC-71 and MHH-ES-1 cells suggests the involvement of the unfolded protein response. While viscum and viscumTT extract treatment indicate response to oxidative stress and activation of stress-mediated MAPK signalling, TT extract treatment suggests the involvement of TLR signalling and autophagy.ConclusionsSince the combinatory extract viscumTT exerts highly effective pro-apoptotic effects on Ewing sarcoma cells in vitro, this phytopolychemotherapy could be a promising adjuvant therapeutic option for paediatric patients with Ewing sarcoma.

Role of spliceosome proteins in the regulation of glucocorticoid receptor isoforms by cortisol and dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) can counteract the activity of cortisol by modulating the glucocorticoid receptor β (GRβ) expression and antagonizing the binding of GRα to the glucocorticoid responsive element (GRE) in RACK1 (Receptor for Activated C Kinase 1) promoter. These observations are important in the context of immunosenescence and can be extended to recognize a complex hormonal balance in the control of GR isoform expression and consequently in the expression of GR responsive genes. To elucidate the mechanism of DHEA on GR alternative splicing, we investigated its possible involvement in the expression of proteins such as the Serine/arginine (SR)-Rich Splicing Factors (SRSF) regulating GR splicing, specifically SRSF9 and SRSF3 also known as SRp30c and SRp20 respectively. We demonstrated that DHEA can induce the up-regulation of GR mRNA which is preferentially directed toward the β isoform. The effect is due to an increase in expression of the splicing factor SRSF9. On the other hand cortisol up-regulated SRSF3, the splicing factor promoting GRα isoform. We demonstrated that DHEA and cortisol modulate SRSF9 and SRSF3 in a different way and our data suggest that the anti-glucocorticoid effect of DHEA, among other mechanisms, is also exerted by modulating the expression of proteins involved in the splicing of the GR pre-mRNA.

SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing.

Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine. Previous work from our laboratory revealed the splicing factor SRSF1 as a regulator of the SUMO pathway, leading us to explore a connection between this pathway and the splicing machinery. We show here that addition of a recombinant SUMO-protease decreases the efficiency of pre-mRNA splicing in vitro. By mass spectrometry analysis of anti-SUMO immunoprecipitated proteins obtained from purified splicing complexes formed along the splicing reaction, we identified spliceosome-associated SUMO substrates. After corroborating SUMOylation of Prp3 in cultured cells, we defined Lys 289 and Lys 559 as bona fide SUMO attachment sites within this spliceosomal protein. We further demonstrated that a Prp3 SUMOylation-deficient mutant while still capable of interacting with U4/U6 snRNP components, is unable to co-precipitate U2 and U5 snRNA and the spliceosomal proteins U2-SF3a120 and U5-Snu114. This SUMOylation-deficient mutant fails to restore the splicing of different pre-mRNAs to the levels achieved by the wild type protein, when transfected into Prp3-depleted cultured cells. This mutant also shows a diminished recruitment to active spliceosomes, compared to the wild type protein. These findings indicate that SUMO conjugation plays a role during the splicing process and suggest the involvement of Prp3 SUMOylation in U4/U6•U5 tri-snRNP formation and/or recruitment.

Crystal structure of U2 snRNP SF3b components: Hsh49p in complex with Cus1p-binding domain

Spliceosomal proteins Hsh49p and Cus1p are components of SF3b, which together with SF3a, Msl1p/Lea1p, Sm proteins, and U2 snRNA, form U2 snRNP, which plays a crucial role in pre-mRNA splicing. Hsh49p, comprising two RRMs, forms a heterodimer with Cus1p. We determined the crystal structures of Saccharomyces cerevisiae full-length Hsh49p as well as its RRM1 in complex with a minimal binding region of Cus1p (residues 290–368). The structures show that the Cus1 fragment binds to the α-helical surface of Hsh49p RRM1, opposite the four-stranded β-sheet, leaving the canonical RNA-binding surface available to bind RNA. Hsh49p binds the 5′ end region of U2 snRNA via RRM1. Its affinity is increased in complex with Cus1(290-368)p, partly because an extended RNA-binding surface forms across the protein–protein interface. The Hsh49p RRM1–Cus1(290-368)p structure fits well into cryo-EM density of the Bact spliceosome, corroborating the biological relevance of our crystal structure.

TDRD6 mediates early steps of spliceosome maturation in primary spermatocytes.

Tudor containing protein 6 (TDRD6) is a male germ line-specific protein essential for chromatoid body (ChB) structure, elongated spermatid development and male fertility. Here we show that in meiotic prophase I spermatocytes TDRD6 interacts with the key protein arginine methyl transferase PRMT5, which supports splicing. TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA and in an arginine methylation dependent manner. In Tdrd6-/- diplotene spermatocytes PRMT5 association with SmB and arginine dimethylation of SmB are much reduced. TDRD6 deficiency impairs the assembly of spliceosomes, which feature 3.5-fold increased levels of U5 snRNPs. In the nucleus, these deficiencies in spliceosome maturation correlate with decreased numbers of SMN-positive bodies and Cajal bodies involved in nuclear snRNP maturation. Transcriptome analysis of TDRD6-deficient diplotene spermatocytes revealed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions. Together, this study demonstrates a novel function of TDRD6 in spliceosome maturation and mRNA splicing in prophase I spermatocytes.

Diabetic polyneuropathy, sensory neurons, nuclear structure and spliceosome alterations: a role for CWC22.

ABSTRACTUnique deficits in the function of adult sensory neurons as part of their early neurodegeneration might account for progressive polyneuropathy during chronic diabetes mellitus. Here, we provide structural and functional evidence for aberrant pre-mRNA splicing in a chronic type 1 model of experimental diabetic polyneuropathy (DPN). Cajal bodies (CBs), unique nuclear substructures involved in RNA splicing, increased in number in diabetic sensory neurons, but their expected colocalization with survival motor neuron (SMN) proteins was reduced – a mislocalization described in motor neurons of spinal muscular atrophy. Small nuclear ribonucleoprotein particles (snRNPs), also participants in the spliceosome, had abnormal multiple nuclear foci unassociated with CBs, and their associated snRNAs were reduced. CWC22, a key spliceosome protein, was aberrantly upregulated in diabetic dorsal root ganglia (DRG), and impaired neuronal function. CWC22 attenuated sensory neuron plasticity, with knockdown in vitro enhancing their neurite outgrowth. Further, axonal delivery of CWC22 siRNA unilaterally to locally knock down the aberrant protein in diabetic nerves improved aspects of sensory function in diabetic mice. Collectively, our findings identify subtle but significant alterations in spliceosome structure and function, including dysregulated CBs and CWC22 overexpression, in diabetic sensory neurons that offer new ideas regarding diabetic sensory neurodegeneration in polyneuropathy.

Quantitative proteomic analysis of human testis reveals system-wide molecular and cellular pathways associated with non-obstructive azoospermia

Male infertility accounts for half of the infertility problems experienced by couples. Azoospermia, having no measurable level of sperm in seminal fluid, is one of the known conditions resulting in male infertility. In order to elucidate the complex molecular mechanisms causing male azoospermia, label-free quantitative shotgun proteomics was carried out on testicular tissue specimens from patients with obstructive azoospermia and non-obstructive azoospermia, including maturation arrest (MA) and Sertoli cell only syndrome (SCOS). The abundance of 520 proteins was significantly changed across three groups of samples. We were able to identify several functional biological pathways enriched in azoospermia samples and confirm selected differentially abundant proteins, using multiple histological methods. The results revealed that cell cycle and proteolysis, and RNA splicing were the most significant biological processes impaired by the substantial suppression of proteins related to the aforementioned categories in SCOS tissues. In the MA patient testes, generation of precursor metabolites and energy as well as oxidation-reduction were the most significantly altered processes. Novel candidate proteins identified in this study include key transcription factors, many of which have not previously been shown to be associated with azoospermia. Our findings can provide substantial insights into the molecular regulation of spermatogenesis and human reproduction. SignificanceThe obtained data showed a drastic suppression of proteins involved in spliceosome, cell cycle and proteasome proteins, as well as energy and metabolic production in Sertoli cell only syndrome testis tissue, and to a lesser extent in maturation arrest samples. Moreover, we identified new transcription factors that are highly down-regulated in SCOS and MA patients, thus helping to understand the molecular complexity of spermatogenesis in male infertility. Our findings provide novel candidate protein targets associated with SCOS or MA azoospermia.

Aberrant RNA splicing and mutations in spliceosome complex in acute myeloid leukemia.

The spliceosome, the cellular splicing machinery, regulates RNA splicing of messenger RNA precursors (pre-mRNAs) into maturation of protein coding RNAs. Recurrent mutations and copy number changes in genes encoding spliceosomal proteins and splicing regulatory factors have tumor promoting or suppressive functions in hematological malignancies, as well as some other cancers. Leukemia stem cell (LSC) populations, although rare, are essential contributors of treatment failure and relapse. Recent researches have provided the compelling evidence that link the erratic spicing activity to the LSC phenotype in acute myeloid leukemia (AML). In this article, we describe the diverse roles of aberrant splicing in hematological malignancies, particularly in AML and their contributions to the characteristics of LSC. We review these promising strategies to exploit the addiction of aberrant spliceosomal machinery for anti-leukemic therapy with aim to eradicate LSC. However, given the complexity and plasticity of spliceosome and not fully known functions of splicing in cancer, the challenges facing the development of the therapeutic strategies targeting RAN splicing are highlighted and future directions are discussed too.

Spliceosomal U1A Protein-SL2 RNA Binding Affinity Decreases in Cells

While extensive biochemical and biophysical studies have been carried out to elucidate protein-RNA binding mechanisms and dynamics in vitro, most of these studies do not take into consideration the effect of the cellular environment. Here we have experimentally tested the role of the cellular environment on protein transport and binding affinity in one of the most widely studied RNA recognition motifs, the spliceosomal protein U1A, and its binding partner, stem loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and binding kinetics were monitored in live U2OS cells by fast relaxation imaging (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 RNA mediates transport of the U1A protein to the nucleus. The binding affinity in live cells was reduced compared to in vitro. The dissociation rate was unchanged in cells; however, the association rate was an order of magnitude lower, resulting in a two order of magnitude decrease in the dissociation constant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further stabilized the complex. This suggests that differences between binding affinities as measured in vitro and in live cells cannot be explained by crowding alone. Instead, high binding affinities as measured in vitro may be necessary for selectivity in vivo, where competition exists between multiple binding partners.

Molecular Pathways: Understanding and Targeting Mutant Spliceosomal Proteins.

Splicing of precursor messenger RNA is a critical step in regulating gene expression, and major advances are being made in understanding the composition and structure of the enzymatic complex that performs splicing, which is termed the "spliceosome." In parallel, there has been increased appreciation for diverse mechanisms by which alterations in splicing contribute to cancer pathogenesis. Key among these include change-of-function mutations in genes encoding spliceosomal proteins. Such mutations are among the most common genetic alterations in myeloid and lymphoid leukemias, making efforts to therapeutically target cells bearing these mutations critical. To this end, recent studies have clarified that pharmacologic modulation of splicing may be preferentially lethal for cells bearing spliceosomal mutations and may also have a role in the therapy of MYC-driven cancers. This has culminated in the initiation of a clinical trial of a novel oral spliceosome modulatory compound targeting the SF3B complex, and several novel alternative approaches to target splicing are in development as reviewed here. There is now, therefore, a great need to understand the mechanistic basis of altered spliceosomal function in cancers and to study the effects of spliceosomal modulatory compounds in preclinical settings and in well-designed clinical trials. Clin Cancer Res; 23(2); 336-41. ©2016 AACR.

Mutations in the Splicosome Lead to Alternative Splicing of CSF3R in Promoting Myelodysplastic Syndromes

Despite advances into the genetic basis of myelodysplastic syndromes (MDS), the mechanisms of their pathophysiology remain poorly understood, and frequently terminates in acute myeloid leukemia. Mutually exclusive mutations have been identified in splicing factors U2AF1, SF3B1, SRSF2, SF3A1, ZRSR2 and LUcL7 in 85% of MDS cases. We and others have identified aberrant forms of Granulocyte Colony Stimulating Factor Receptor (CSF3R) in patients with myeloid malignancies and partially characterized their phenotypic and signal transduction effects. Two of the seven isoforms of CSF3R have clinical relevance: Class I (full-length form) and Class IV (alternatively spliced form). Compared to Class I, Class IV isoform is differentiation defective and displays enhanced proliferation. Increased expression of the Class IV isoform has been found in MDS/AML and results in enhanced cell proliferation and impaired differentiation of granulocytes (Mehta et al, Leukemia 2014). The Class IV CSF3R transcript is derived by intron excision, whereas Class I results from retention of that intron. We hypothesize aberrant splicing of the CSF3R transcript promoted by mutations in splicing factors drives abnormal granulopoiesis in MDS due to increased expression of the differentiation defective Class IV CSF3R.We identified U2AF1, SRSF2, and SAM68 sites within the nucleotide sequence of the spliced intron. To study the effect of splicing factor mutations on CSF3R splicing, we constructed a minigene consisting of 5’ exon of the CAT gene with an ATG site and a partial intron fused to intron region of G-CSFR upstream of Exon 17. The retained intron is present in the exon 17 of CSF3R. The minigene consists of 2 introns and 3 exons. We transduced 293FT cells with the minigene ± wild-type U2AF1 or U2AF1 S34F mutant, as well as the minigene ± wild-type SF3B1 or SF3B1 K300E. The cells were then treated with either 1µM sodium orthovanadate (Na3VO4) or 10 nM phorbol myristic acetate (PMA). The former inhibits tyrosine phosphatases, promoting tyrosine protein phosphorylation, whereas the latter promotes activation of the protein kinase C pathway, which eventually activates ERK1/2 an upstream activator of SAM68. Protein phosphorylation of spliceosome proteins is fundamental to regulation of splicing. The effect of PMA and Na3VO4 were compared with each other and those treated with control diluent. Qualitative and semi-quantitative evaluations were performed. We identified expression with SF3B1 K700E enhances CSF3R splicing resulting in increased Class IV, however the U2AF1 S34F inhibits splicing resulting in reduced Class IV. The increase and decrease were in relation to the wild type. Further exploration of how cellular protein phosphorylation influences splicing using Na3VO4 (identified that cells transduced with SF3B1, U2AF1 wt or mutant shown decreased splicing resulting in Class IV transcript. PMA however had no effect on either class expression. These data suggest that CSF3R transcript processing is sensitive to diverse tyrosine kinase signaling, resulting in increased intron retention, suggesting that phosphorylation events inhibit Class IV transcript formation. Additionally the mutations in splicing factors which are recurrently found in MDS are associated with altered CSF3R transcripts and may affect CSF3R signaling and cell phenotype.(This work is supported by DOD and NIH R01R01HL128173.) DisclosuresNo relevant conflicts of interest to declare.

Mutations in the Spliceosome Lead to Alternative Splicing of CSF3R in Promoting Myelodysplastic Syndromes

Introduction: Despite advances into the genetic basis of myelodysplastic syndromes (MDS), the mechanisms of their pathophysiology remain poorly understood, and frequently terminates in acute myeloid leukemia. Mutually exclusive mutations have been identified in splicing factors U2AF1, SF3B1, SRSF2, SF3A1, ZRSR2 and LUcL7 in 85% of MDS cases. We and others have identified aberrant forms of Granulocyte Colony Stimulating Factor Receptor (CSF3R) in patients with myeloid malignancies and partially characterized their phenotypic and signal transduction effects. Two of the seven isoforms of CSF3R have clinical relevance: Class I (full-length form) and Class IV (alternatively spliced form). Compared to Class I, Class IV isoform is differentiation defective and displays enhanced proliferation. Increased expression of the Class IV isoform has been found in MDS/AML and results in enhanced cell proliferation and impaired differentiation of granulocytes (Mehta et al, Leukemia 2014). The Class IV CSF3R transcript is derived by intron excision, whereas Class I results from retention of that intron. We hypothesize aberrant splicing of the CSF3R transcript promoted by mutations in splicing factors drives abnormal granulopoiesis in MDS due to increased expression of the differentiation defective Class IV CSF3R.Methods: We identified U2AF1, SRSF2, and SAM68 sites within the nucleotide sequence of the spliced intron. To study the effect of splicing factor mutations on CSF3R splicing, we constructed a minigene consisting of 5’ exon of the CAT gene with an ATG site and a partial intron fused to intron region of G-CSFR upstream of Exon 17. The retained intron is present in the exon 17 of CSF3R. The minigene consists of 2 introns and 3 exons. We transduced 293FT cells with the minigene ± wild-type U2AF1 or U2AF1 S34F mutant, as well as the minigene ± wild-type SF3B1 or SF3B1 K300E. The cells were then treated with either 1µM sodium orthovanadate (Na3VO4) or 10 nM phorbol myristic acetate (PMA). The former inhibits tyrosine phosphatases, promoting tyrosine protein phosphorylation, whereas the latter promotes activation of the protein kinase C pathway, which eventually activates ERK1/2 an upstream activator of SAM68. Protein phosphorylation of spliceosome proteins is fundamental to regulation of splicing. The effect of PMA and Na3VO4 were compared with each other and those treated with control diluent. Qualitative and semi-quantitative evaluations were performed.Results: We identified expression with SF3B1 K700E enhances CSF3R splicing resulting in increased Class IV, however the U2AF1 S34F inhibits splicing resulting in reduced Class IV. The increase and decrease were in relation to the wild type. Further exploration of how cellular protein phosphorylation influences splicing using Na3VO4 (identified that cells transduced with SF3B1, U2AF1 wt or mutant shown decreased splicing resulting in Class IV transcript. PMA however had no effect on either class expression.Conclusions: These data suggest that CSF3R transcript processing is sensitive to diverse tyrosine kinase signaling, resulting in increased intron retention, suggesting that phosphorylation events inhibit Class IV transcript formation. Additionally, the mutations in splicing factors which are recurrently found in MDS are associated with altered CSF3Rtranscripts and may affect CSF3R signaling and cell phenotype.This work is supported by DOD Bone Marrow Failure Idea Award and NIH R01HL128173. DisclosuresNo relevant conflicts of interest to declare.

Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment.

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598–631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of β1‐α1‐β2‐β3‐α2‐β4. Furthermore, a docking model based on NOESY measurements suggests that residues 607–616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix–helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull‐down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix–helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145.

Spliceosomal protein eftud2 mutation leads to p53-dependent apoptosis in zebrafish neural progenitors.

Haploinsufficiency of EFTUD2 (Elongation Factor Tu GTP Binding Domain Containing 2) is linked to human mandibulofacial dysostosis, Guion-Almeida type (MFDGA), but the underlying cellular and molecular mechanisms remain to be addressed. We report here the isolation, cloning and functional analysis of the mutated eftud2 (snu114) in a novel neuronal mutant fn10a in zebrafish. This mutant displayed abnormal brain development with evident neuronal apoptosis while the development of other organs appeared less affected. Positional cloning revealed a nonsense mutation such that the mutant eftud2 mRNA encoded a truncated Eftud2 protein and was subjected to nonsense-mediated decay. Disruption of eftud2 led to increased apoptosis and mitosis of neural progenitors while it had little effect on differentiated neurons. Further RNA-seq and functional analyses revealed a transcriptome-wide RNA splicing deficiency and a large amount of intron-retaining and exon-skipping transcripts, which resulted in inadequate nonsense-mediated RNA decay and activation of the p53 pathway in fn10a mutants. Therefore, our study has established that eftud2 functions in RNA splicing during neural development and provides a suitable zebrafish model for studying the molecular pathology of the neurological disease MFDGA.

Scaffolding in the Spliceosome via Single α Helices

The spliceosomal B complex-specific protein Prp38 forms a complex with the intrinsically unstructured proteins MFAP1 and Snu23. Our binding and crystal structure analyses show that MFAP1 and Snu23 contact Prp38 via ER/K motif-stabilized single α helices, which have previously been recognized only as rigidconnectors or force springs between protein domains. A variant of the Prp38-binding single α helix of MFAP1, in which ER/K motifs not involved in Prp38 binding were mutated, was less α-helical in isolation and showed a reduced Prp38 affinity, with opposing tendencies in interaction enthalpy and entropy. Our results indicate that the strengths of single α helix-based interactions can be tuned by the degree of helix stabilization in the unbound state. MFAP1, Snu23, and several other spliceosomal proteins contain multiple regions that likely form single α helices via which they might tether several binding partners and act as intermittent scaffolds that facilitate remodeling steps during assembly of an active spliceosome.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.

Therapeutic targeting of splicing in cancer.

Recent studies have highlighted that splicing patterns are frequently altered in cancer and that mutations in genes encoding spliceosomal proteins, as well as mutations affecting the splicing of key cancer-associated genes, are enriched in cancer. In parallel, there is also accumulating evidence that several molecular subtypes of cancer are highly dependent on splicing function for cell survival. These findings have resulted in a growing interest in targeting splicing catalysis, splicing regulatory proteins, and/or specific key altered splicing events in the treatment of cancer. Here we present strategies that exist and that are in development to target altered dependency on the spliceosome, as well as aberrant splicing, in cancer. These include drugs to target global splicing in cancer subtypes that are preferentially dependent on wild-type splicing for survival, methods to alter post-translational modifications of splicing-regulating proteins, and strategies to modulate pathologic splicing events and protein-RNA interactions in cancer.

Tau mis-splicing in the pathogenesis of neurodegenerative disorders.

Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders. [BMB Reports 2016; 49(8): 405-413]

Resistance to imatinib in patients with chronic myelogenous leukemia and the splice variant BCR-ABL135INS

PurposeIn patients with chronic myelogenous leukemia (CML), point mutations in the BCR-ABL1 kinase domain are the most common cause of treatment failure with a tyrosine kinase inhibitor (TKI). It is not clear whether the splice variant BCR-ABL135INS is also associated with treatment failure. Patients and methodsWe reviewed all CML patients who had BCR-ABL1 kinase mutation analysis performed between August 1, 2007, and January 15, 2014. Patients who had BCR-ABL135INS detected had their medical records reviewed to determine response to TKI therapy. ResultsTwo hundred and eighty four patients had kinase mutation testing performed; of these, 64 patients (23%) had BCR-ABL135INS detected. Forty-five patients were in chronic phase (70%), 10 were in accelerated phase (16%), 6 were in blastic phase (9%), and 3 were in other settings (5%). Of the 34 chronic phase patients who began therapy with imatinib, 23 patients (68%) failed therapy: 8 patients (24%) had primary refractory disease, 11 patients (32%) progressed, and 4 patients (12%) had disease progression after dose interruption. In contrast to the patients with disease progression or lack of response, none of 23 patients who were responding to imatinib had BCR-ABL135INS detected. DNA sequencing of commonly mutated spliceosomal genes SF3B1, U2AF1, SRSF2, ZRSR2, SFA31, PRPF408, U2A565, and SF1 did not reveal mutations in seven BCR-ABL135INS –positive patients tested. ConclusionsThe splice variant BCR-ABL135INS is frequently found in patients who are resistant to imatinib. Mutations in the commonly mutated spliceosomal proteins do not contribute to this association.