Search

Aggregation of the microtubule-associated protein tau characterizes tauopathies, including Alzheimer's disease and frontotemporal lobar degeneration (FTLD-Tau). Gene expression regulation of tau is complex and incompletely understood. Here we report that the human tau gene (MAPT) generates two circular RNAs (circRNAs) through backsplicing of exon 12 to either exon 7 (12→7 circRNA) or exon 10 (12→10 circRNA). Both circRNAs lack stop codons. The 12→7 circRNA contains one start codon and is translated in a rolling circle, generating a protein consisting of multimers of the microtubule-binding repeats R1–R4. For the 12→10 circRNA, a start codon can be introduced by two FTLD-Tau mutations, generating a protein consisting of multimers of the microtubule-binding repeats R2–R4, suggesting that mutations causing FTLD may act in part through tau circRNAs. Adenosine to inosine RNA editing dramatically increases translation of circRNAs and, in the 12→10 circRNA, RNA editing generates a translational start codon by changing AUA to AUI. Circular tau proteins self-aggregate and promote aggregation of linear tau proteins. Our data indicate that adenosine to inosine RNA editing initiates translation of human circular tau RNAs, which may contribute to tauopathies.

The mechanisms through which oral commensal bacteria mitigates uncontrolled inflammatory responses of the oral mucosa remain unknown. Here, we show that representative oral bacterial species normally associated with oral health [S. gordonii (Sg), V. parvula (Vp), A. naeslundii (An), C. sputigena (Cs), and N. mucosa (Nm)] enhanced differential chemokine responses in oral epithelial cells (OECs), with some bacteria (An, Vp, and Nm) inducing higher chemokine levels (CXCL1, CXCL8) than others (Sg, Cs). Although all bacterial species (except Cs) increased CCL20 mRNA levels consistent with protein elevations in cell lysates, only An, Vp, and Nm induced higher CCL20 secretion, similar to the effect of the oral pathogen F. nucleatum (Fn). In contrast, most CCL20 remained associated with OECs exposed to Sg and negligible amounts released into the cell supernatants. Consistently, Sg attenuated An-induced CCL20. MiR-4516 and miR-663a were identified as Sg-specifically induced miRNAs modulating validated targets of chemokine-associated pathways. Cell transfection with miR-4516 and miR-663a decreased An- and Fn-induced CCL20. MiRNA upregulation and attenuation of An-induced CCL20 by Sg were reversed by catalase. Up-regulation of both miRNAs was specifically enhanced by oral streptococci H(2)O(2)-producers. These findings suggest that CCL20 levels produced by OECs in response to bacterial challenge are regulated by Sg-induced miR-4516 and miR-663a in a mechanism that involves hydrogen peroxide. This type of molecular mechanism could partly explain the central role of specific oral streptococcal species in balancing inflammatory and antimicrobial responses given the critical role of CCL20 in innate (antimicrobial) and adaptive immunity (modulates Th17 responses).

C/D box snoRNAs (SNORDs) are a highly expressed class of non-coding RNAs. Besides their well-established role in rRNA modification, C/D box snoRNAs form protein complexes devoid of fibrillarin and regulate pre-mRNA splicing and polyadenylation of numerous genes. There is an emerging body of evidence for functional interactions between RNA viruses and C/D box snoRNAs. The infectivity of some RNA viruses depends on enzymatically active fibrillarin, and many RNA viral proteins associate with nucleolin or nucleophosmin, suggesting that viruses benefit from their cytosolic accumulation. These interactions are likely reflected by morphological changes in the nucleolus, often leading to relocalization of nucleolar proteins and ncRNAs to the cytosol that are a characteristic feature of viral infections. Knock-down studies have also shown that RNA viruses need specific C/D box snoRNAs for optimal replication, suggesting that RNA viruses benefit from gene expression programs regulated by SNORDs, or that viruses have evolved “new” uses for these humble ncRNAs to advance their prospects during infection.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5' splice site with an upstream 3' splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5' splice site with an upstream 3' splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Background: Microsurgery of larynx requires free access to the operation field. It is possible under general anaesthesia with high frequency jet ventilation. Shared upper airway demands anaesthesiologist and surgeon cooperation. Methods: 40 patients scheduled for laser CO2 microsurgery were included in the study. 20 of patients presented malignant pathology and 20 benign lesions. Anaesthetic risk was assessed in ASA-scale. Patients under 18 and above 80 years old, with severe COPD and renal or hepatic failure were excluded. This article deals with preoperative assessment and management of general anaesthesia. Conditions for intubation were assessed using Krieg-score and conditions for surgery were assessed by surgeon using Likert-scale. Results: Presented method of anaesthesia enables efficient and safe surgical treatment. Conditions for surgery were highly scored by surgeon. Discussion: High risk of complications is associated with microsurgery of larynx under general anaesthesia with jet ventilation. Close cooperation between surgical and anaesthetic teams is essential for safety of this procedure.

The microtubule-associated protein Tau, generated by the MAPT gene is involved in dozens of neurodegenerative conditions (“tauopathies”), including Alzheimer's disease (AD) and frontotemporal lobar degeneration/frontotemporal dementia (FTLD/FTD). The pre-mRNA of MAPT is well studied and its aberrant pre-mRNA splicing is associated with frontotemporal dementia. Using a PCR screen of RNA from human brain tissues, we found that the MAPT locus generates circular RNAs through a backsplicing mechanism from exon 12 to either exon 10 or 7. MAPT circular RNAs are localized in the cytosol and contain open reading frames encoding Tau protein fragments. The MAPT exon 10 is alternatively spliced and proteins involved in its regulation, such as CLK2, SRSF7/9G8, PP1 (protein phosphatase 1) and NIPP1 (nuclear inhibitor of PP1) reduce the abundance of the circular MAPT exon 12 → 10 backsplice RNA after being transfected into cultured HEK293 cells. In summary, we report the identification of new bona fide human brain RNAs produced from the MAPT locus. These may be a component of normal human brain Tau regulation and, since the circular RNAs could generate high molecular weight proteins with multiple microtubule binding sites, they could contribute to taupathies.

The serotonin receptor 2C (5HT2C) is an important drug target to treat obesity and depression. Its pre-mRNA undergoes alternative splicing, encoding a short RNA1 isoform that is localized intracellularly and a full-length isoform (RNA2) that can reach the cell membrane. These splicing isoforms are deregulated in Prader-Willi syndrome (PWS), due to the loss of a trans-acting regulatory RNA, SNORD115. Here we show that the 5HT2C mRNA is expressed in the posterior pituitary, suggesting that 5HT2C mRNA is generated in the hypothalamus and subsequently conveyed by axonal transport. In the pituitary, the ratio of 5HT2C isoforms is regulated by feeding, and can be manipulated using a splice-site changing oligonucleotide injected into the blood. The pituitary expression of the 5HT2C mRNA may constitute a previously unknown mechanism whereby serotonin in the circulation or drugs targeting the 5HT2C might induce side-effects. Finally, the deregulation of 5HT2C splicing isoforms in PWS could contribute to the known hormonal imbalances.

Oncogenic metadherin is a key contributor to tumourigenesis with metadherin expression and cytoplasmic localisation previously linked to poor survival. A number of reports have shown metadherin localises specifically to nuclear speckles known to be rich in RNA-binding proteins including the splicing proteins YTHDC1, Sam68 and T-STAR, that have been shown to select alternative splice sites in mRNA of tumour-associated proteins including BRCA, MDM2 and VEGF. Here we investigate the interaction and relationship between metadherin and the splice factors YTHDC1, T-STAR and Sam68. Using a yeast two-hybrid assay and immunoprecipitation we show that metadherin interacts with YTHDC1, Sam68 and T-STAR and demonstrate that T-STAR is significantly overexpressed in prostate cancer tissue compared to benign prostate tissue. We also demonstrate that metadherin influences splice site selection in a dose-dependent manner in CD44v5-luc minigene reporter assays. Finally, we demonstrate that prostate cancer patients with higher metadherin expression have greater expression of the CD44v5 exon. CD44v5 expression could be used to discriminate patients with poor outcomes following radical prostatectomy. In this work we show for the first time that metadherin interacts with, and modulates, the function of key components of splicing associated with cancer development and progression.

Circular RNAs are a recently discovered class of RNAs formed by covalently linking the 5' and 3' end of an RNA. Pre-mRNAs generate circular RNAs through a back-splicing mechanism. Whereas in linear splicing a 5' splice site is connected to a downstream 3' splice site, in back-splicing the 5' splice site is connected to an upstream 3' splice site. Both mechanisms use the spliceosome for catalysis. For back-splicing to occur, the back-splice sites must frequently be brought into close proximity, which is achieved through the formation of secondary structures in the pre-mRNA. In general, these pre-mRNA structures are formed by RNA base pairing between complementary sequences flanking the back-splicing sites. Proteins can abolish these RNA structures through binding to one of the complementary strands. However, proteins can also promote back-splicing without strong RNA structures through multimerization after binding to intronic regions flanking circular exons. In humans, Alu-elements comprising around 11% of the human genome are the best-characterized elements generating structures promoting circular RNA formation. Thus, intronic pre-mRNA structures contribute to the formation of circular RNAs.

Almost all human protein coding genes undergo alternative splicing, and an increasing number of diseases is associated with the selection of ‘wrong’ splice sites. Alternative missplicing can be caused by deoxyribonucleic acid point mutations, changes in repetitive sequences or alterations in proteins that regulate splicing. The effect of these mutations can be evaluated by bioinformatic means, but these predictions need to be verified experimentally. With the exception of premature stop codons that are introduced by aberrantly spliced exons, the mechanism of pathological exon usage that leads to a disease is generally poorly understood. A pathological change in the activity of splicing factors typically results in numerous small changes in exon usage. Numerous screening efforts identified oligonucleotides and small molecular weight substances that can be tested as therapeutic approaches for diseases caused by missplicing. Key Concepts: Alternative splice site selection is regulated by combinatorial control through a network of protein–protein, protein–RNA and RNA–RNA interactions. Mutations in the splice sites, exonic and intronic sequences lead to aberrant exon usage. Mutations can cause exon skipping, a change in the degree of exon usage or activate sequences on the pre-mRNA to become a new exon. Synonymous mutations in exons can cause their aberrant usage, which can be a disease mechanism. A pathological change in the activity of splicing regulatory proteins results in numerous, individually small changes of alternative exon usage. Aberrant exon usage can be targeted by rationale therapies, several oligonucleotide-based treatments are tested in clinical trials and small molecules were identified that change splice site selection by high-throughput screens. Keywords: alternative splicing; splicing regulatory elements; disease causing mutations; exon enhancer; exon silencer

Interphase chromatin is arranged into topologically separated domains comprising gene expression and replication units through genomic sequence elements, so-called MAR or SAR regions (from matrix- or scaffold-associating regions). S/MAR regions are located near the boundaries of actively transcribed genes and were shown to influence their activity. We show that scaffold attachment factor B (SAF-B), which specifically binds to S/MAR regions, interacts with RNA polymerase II (RNA pol II) and a subset of serine-/arginine-rich RNA processing factors (SR proteins). SAF-B localized to the nucleus in a speckled pattern that conicided with the distribution of the SR protein SC35. Furthermore, we show that overexpressed SAF-B induced an increase of the 10S splice product using an E1A reporter gene and repressed the activity of an S/MAR flanked CAT reporter gene construct in vivo. This indicates an association of SAF-B with SR proteins and components of the transcription machinery. Our results describe the coupling of a chromatin organizing S/MAR element with transcription and pre-mRNA processing components and we propose that SAF-B serves as a molecular base to assemble a "transcriptosome complex" in the vicinity of actively transcribed genes.

The central nervous system-specific serotonin receptor 2C (5HT2C) controls key physiological functions, such as food intake, anxiety, and motoneuron activity. Its deregulation is involved in depression, suicidal behavior, and spasticity, making it the target for antipsychotic drugs, appetite controlling substances, and possibly anti-spasm agents. Through alternative pre-mRNA splicing and RNA editing, the 5HT2C gene generates at least 33 mRNA isoforms encoding 25 proteins. The 5HT2C is a G-protein coupled receptor that signals through phospholipase C, influencing the expression of immediate/early genes like c-fos. Most 5HT2C isoforms show constitutive activity, i.e., signal without ligand binding. The constitutive activity of 5HT2C is decreased by pre-mRNA editing as well as alternative pre-mRNA splicing, which generates a truncated isoform that switches off 5HT2C receptor activity through heterodimerization; showing that RNA processing regulates the constitutive activity of the 5HT2C system. RNA processing events influencing the constitutive activity target exon Vb that forms a stable double stranded RNA structure with its downstream intron. This structure can be targeted by small molecules and oligonucleotides that change exon Vb alternative splicing and influence 5HT2C signaling in mouse models, leading to a reduction in food intake. Thus, the 5HT2C system is a candidate for RNA therapy in multiple models of CNS disorders.

Mutations in the RNA binding protein FUS (fused in sarcoma) have been linked to a subset of familial amyotrophic lateral sclerosis (ALS) cases. The mutations are clustered in the C-terminal nuclear localization sequence (NLS). Various FUS mutants accumulate in the cytoplasm whereas wild-type (WT) FUS is mainly nuclear. Here we investigate the effect of one ALS causing mutant (FUS-ΔNLS, also known as R495X) on pre-mRNA splicing and RNA expression using genome wide exon-junction arrays. Using a non-neuronal stable cell line with inducible FUS expression, we detected early changes in RNA composition. In particular, mutant FUS-ΔNLS increased calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2) at both mRNA and protein levels, whereas WT-FUS had no effect. Chromatin immunoprecipitation experiments showed that FUS-ΔNLS accumulated at the CAMK2N2 promoter region, whereas promoter occupation by WT-FUS remained constant. Given the loss of FUS-ΔNLS in the nucleus through the mutation-induced translocation, this increase of promoter occupancy is surprising. It indicates that, despite the obvious cytoplasmic accumulation, FUS-ΔNLS can act through a nuclear gain of function mechanism.

The ubiquitously expressed splicing factor YT521 (YTHDC1) is characterized by alternatively spliced isoforms with regulatory impact on cancer-associated gene expression. Our recent findings account for the prognostic significance of YT521 in endometrial cancer. In this study, we investigated the hypoxia-dependency of YT521 expression as well as its differential isoform activities on oncological important target genes. YT521's potential regulatory influence on splicing was investigated by a minigene assay for the specific target gene CD44. Functional splicing analysis was performed by YT521 knock-down or overexpression, respectively. In addition, YT521 expression was determined under hypoxia. The two protein-generating YT521 mRNA isoforms 1 and 2 caused a comparable, specific induction of CD44v alternative splicing (P

Prader-Willi syndrome (PWS) is caused by the loss of RNA expression from an imprinted region on chromosome 15 that includes SNRPN, SNORD115, and SNORD116. Currently, there are no mouse models that faithfully reflect the human phenotype and investigations rely on human post-mortem material. During molecular characterization of tissue deposited in a public brain bank from a patient diagnosed with Prader-Willi syndrome, we found RNA expression from SNRPN, SNORD115, and SNORD116 which does not support a genetic diagnosis of Prader-Willi syndrome. The patient was a female, Caucasian nursing home resident with history of morbid obesity (BMI 56.3) and mental retardation. She died at age of 56 from pulmonary embolism. SNORD115 and SNORD116 are unexpectedly stable in post mortem tissue and can be used for post-mortem diagnosis. Molecular characterization of PWS tissue donors can confirm the diagnosis and identify those patients that have been misdiagnosed.

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes premRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2 0 -hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5 0 -splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates >300 alternative exons that form secondary structures enriched in A–U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch.

Significance C/D box small nucleolar RNAs (SNORDs) are abundant, short, nucleoli-residing, noncoding RNAs that guide the methyltransferase fibrillarin to perform 2′- O -methylation of target RNAs. We identified 29 SNORDs present in a fibrillarin-containing fraction as well as a fibrillarin-free fraction enriched in spliceosomes. One of these SNORDs, SNORD27 , directs rRNA methylation and regulates alternative pre-mRNA splicing (AS) of E2F7 pre-mRNA, a transcriptional repressor of cell cycle-regulated genes. SNORD27 likely regulates E2F7 pre-mRNA AS by masking splice sites through base pairing. This previously unidentified function of SNORDs increases the number of factors regulating AS, a critical step in the expression of the vast majority of human genes, and highlights a potential coupling between AS, cell cycle, proliferation, and ribosome biogenesis.

The glycine receptor-deficient mutant mouse spastic carries a full-length long interspersed nuclear element (LINE1) retrotransposon in intron 6 of the glycine receptor β subunit gene, Glrbspa. The mutation arose in the C57BL/6J strain and is associated with skipping of exon 6 or a combination of the exons 5 and 6, thus resulting in a translational frameshift within the coding regions of the GlyR β subunit. The effect of the Glrbspa LINE1 insertion on pre-mRNA splicing was studied using a minigene approach. Sequence comparison as well as motif prediction and mutational analysis revealed that in addition to the LINE1 insertion the inactivation of an exonic splicing enhancer (ESE) within exon 6 is required for skipping of exon 6. Reconstitution of the ESE by substitution of a single residue was sufficient to prevent exon skipping. In addition to the ESE, two regions within the 5′ and 3′ UTR of the LINE1 were shown to be critical determinants for exon skipping, indicating that LINE1 acts as efficient modifier of subtle endogenous splicing phenotypes. Thus, the spastic allele of the murine glycine receptor β subunit gene is a two-hit mutation, where the hypomorphic alteration in an ESE is amplified by the insertion of a LINE1 element in the adjacent intron. Conversely, the LINE1 effect on splicing may be modulated by individual polymorphisms, depending on the insertional environment within the host genome.

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

Almost every protein-coding gene undergoes pre-mRNA splicing, and the majority of these pre-mRNAs are alternatively spliced. Alternative exon usage is regulated by the transient formation of protein complexes on the pre-mRNA that typically contain heterogeneous nuclear ribonucleoproteins (hnRNPs). Here we characterize hnRNP G, a member of the hnRNP class of proteins. We show that hnRNP G is a nuclear protein that is expressed in different concentrations in various tissues and that interacts with other splicing regulatory proteins. hnRNP G is part of the supraspliceosome, where it regulates alternative splice site selection in a concentration-dependent manner. Its action on alternative exons can occur without a functional RNA-recognition motif by binding to other splicing regulatory proteins. The RNA-recognition motif of hnRNP G binds to a loose consensus sequence containing a CC(A/C) motif, and hnRNP G preferentially regulates alternative exons where this motif is clustered in close proximity. The X-chromosomally encoded hnRNP G regulates different RNAs than its Y-chromosomal paralogue RNA-binding motif protein, Y-linked (RBMY), suggesting that differences in alternative splicing, evoked by the sex-specific expression of hnRNP G and RBMY, could contribute to molecular sex differences in mammals.

CD44 is a cell surface glycoprotein involved in cell–cell and cell–substrate interactions, which may be shed or released into circulation by proteolytic enzymatic mechanisms. Alternative splicing of CD44 and aberrant levels of soluble CD44 variants in the serum of cancer patients have been correlated to tumor progression and metastasis in different tumors including breast cancer. In this study we evaluated the clinical value of CD44 serum levels (sCD44) in patients with primary breast cancer. Concentrations of soluble isoforms sCD44std, sCD44v5 and sCD44v6 were determined with a sensitive ELISA and normalized against the total protein concentration (TP). Pre-operative serum samples from 82 patients and 67 age-matched healthy blood donors were analyzed. The results were correlated to clinico-pathological parameters (tumor size, grading, lymph node metastasis, etc.). In sera of breast cancer patients, we detected elevated concentrations of sCD44v6 (P = 0.0001) and total protein TP (P = 0.0001) in comparison to healthy controls, whereas overall sCD44 (sCD44std) and sCD44v5 did not differ. Patients with sCD44v6-concentrations above the 75%-percentile showed an increased T stage (2.9 cm vs. 1.8 cm) as well as a higher risk for lymph node metastasis (55% vs. 35%). In breast cancer patients with lymph node metastasis the median value of sCD44v6 was significantly higher (P = 0.025) in comparison to patients without lymph node metastasis and healthy controls. Our data suggest an upregulated expression of alternatively spliced soluble CD44 isoforms in breast cancer patients. The specific alterations of certain CD44 isoform concentrations (especially sCD44v6) may reflect disturbances of the nuclear splicing machinery in tumor cells. The clinical significance of our findings are underlined by the positive correlation of elevated sCD44v6 concentrations and lymph node metastases (r s = 0.25).

Specialized chromatin structures such as nucleosomes with specific histone modifications decorate exons in eukaryotic genomes, suggesting a functional connection between chromatin organization and the regulation of pre-mRNA splicing. Through profiling the functional location of Poly (ADP) ribose polymerase, we observed that it is associated with the nucleosomes at exon/intron boundaries of specific genes, suggestive of a role for this enzyme in alternative splicing. Poly (ADP) ribose polymerase has previously been implicated in the PARylation of splicing factors as well as regulation of the histone modification H3K4me3, a mark critical for co-transcriptional splicing. In light of these studies, we hypothesized that interaction of the chromatin-modifying factor, Poly (ADP) ribose polymerase with nucleosomal structures at exon–intron boundaries, might regulate pre-mRNA splicing. Using genome-wide approaches validated by gene-specific assays, we show that depletion of PARP1 or inhibition of its PARylation activity results in changes in alternative splicing of a specific subset of genes. Furthermore, we observed that PARP1 bound to RNA, splicing factors and chromatin, suggesting that Poly (ADP) ribose polymerase serves as a gene regulatory hub to facilitate co-transcriptional splicing. These studies add another function to the multi-functional protein, Poly (ADP) ribose polymerase, and provide a platform for further investigation of this protein’s function in organizing chromatin during gene regulatory processes.

Phosphorylation and dephosphorylation of splicing factors play a key role in pre-mRNA splicing events, and cantharidin and norcantharidin analogs inhibit protein phosphatase-1 (PP1) and change alternative pre-mRNA splicing. Targeted inhibitors capable of selectively inhibiting PP-1 could promote exon 7 inclusion in the survival-of-motorneuron-2 gene (SMN2) and shift the proportion of SMN2 protein from a dysfunctional to a functional form. As a prelude to the development of norcantharidin-tethered oligonucleotide inhibitors, the synthesis a norcantharidin-tethered guanosine was developed in which a suitable tether prevented the undesired cyclization of norcantharidin monoamides to imides and possessed a secondary amine terminus suited to the synthesis of oligonucleotides analogs. Application of this methodology led to the synthesis of a diastereomeric mixture of norcantharidin-tethered guanosines, namely bisammonium (1R,2S,3R,4S)- and (1S,2R,3S,4R)-3-((4-(2-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6- dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl) oxy) oxidophosphoryl) oxy) ethyl)-phenethyl)(methyl)carbamoyl)-7-oxabicyclo[2.2.1] heptane-2-carboxylate, which showed activity in an assay for SMN2 pre-mRNA splicing

Tau is a microtubule-associated protein that is important for establishing and maintaining neuronal morphology. In addition to its role in normal cells, tau protein is involved in many neurodegenerative diseases, e.g. Alzheimer's disease (AD) and frontotemporal dementia, as the main component of intraneuronal aggregates. Alternative splicing of tau gene in the brain can give rise to at least six protein variants. A causative role of skewed tau exon 10 inclusion has been defined in frontotemporal dementia; however, no link was established between the aberrant splicing of tau and AD. Here, we applied a single-molecule-based technology, polymerase colony or polony, to simultaneously monitor tau splicing variant and haplotype profile in sporadic AD and normal brains. We found that the coordinated expression of tau exons 2 and 10 is altered in AD. Additional investigations of cis and trans mechanisms of this observation revealed a decreased protein expression of a known tau splicing factor, htra2-beta-1 in AD, thereby implicating a trans mechanism. Our results demonstrate that dysregulation of combinatorial splicing might serve as a signature for aging-related diseases, and the polony assay could be widely adapted for the study of other tauopathies. Furthermore, splicing-based therapeutics is an emerging area of drug development, and a well-defined and quantitative assay for monitoring single-gene transcriptome will be relevant for such development.

Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.

Alternative splicing is a hallmark of glycoprotein hormone receptor gene regulation, but its molecular mechanism is unknown. The LH receptor (LHR) gene possesses 11 exons, but exon 10 is constitutively skipped in the New World monkey lineage (LHR type 2), whereas it is constitutively spliced in the human (LHR type 1). This study identifies the regulatory elements of exon 10 usage. Sequencing of genomic marmoset DNA revealed that the cryptic LHR exon 10 is highly homologous to exon 10 from other species and displays intact splice sites. Functional studies using a minigene approach excluded the contribution of intronic, marmoset-specific long interspersed nucleotide-1 elements to exon 10 skipping. Sequencing of the genomic regions surrounding exon 10 from several primate lineages, sequence comparisons including the human and mouse LHR gene, revealed the presence of unique nucleotides at 3′-intronic position −19 and −10 and at position +26 within exon 10 of the marmoset LHR. Exon trap experiments and in vitro mutagenesis of these nucleotides resulted in the identification of a composite regulatory element of splicing consisting of cis-acting elements represented by two polypyrimidine tracts and a trans-acting element within exon 10, which affect the secondary RNA structure. Changes within this complex resulted either in constitutive exon inclusion, constitutive skipping, or alternative splicing of exon 10. This work delineates the molecular pathway leading to intronization of exon 10 in the LHR type 2 and reveals, for the first time, the essential function of regulatory and structural elements involved in glycoprotein hormone receptor splicing.

The loss of two gene clusters encoding small nucleolar RNAs, SNORD115 and SNORD116 contributes to Prader-Willi syndrome (PWS), the most common syndromic form of obesity in humans. SNORD115 and SNORD116 are considered to be orphan C/D box snoRNAs (SNORDs) as they do not target rRNAs or snRNAs. SNORD115 exhibits sequence complementarity towards the serotonin receptor 2C, but SNORD116 shows no extended complementarities to known RNAs. To identify molecular targets, we performed genome-wide array analysis after overexpressing SNORD115 and SNORD116 in HEK 293T cells, either alone or together. We found that SNORD116 changes the expression of over 200 genes. SNORD116 mainly changed mRNA expression levels. Surprisingly, we found that SNORD115 changes SNORD116’s influence on gene expression. In similar experiments, we compared gene expression in post-mortem hypothalamus between individuals with PWS and aged-matched controls. The synopsis of these experiments resulted in 23 genes whose expression levels were influenced by SNORD116. Together our results indicate that SNORD115 and SNORD116 influence expression levels of multiple genes and modify each other activity.

The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2β3), RS1 blocks phosphorylation of these serines in the full-length Tra2β1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2β1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2β1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis. Background: Serine-arginine-rich (SR)-like proteins regulate the alternative splicing of human genes. Results: The serine-arginine protein kinase 1 (SRPK1) phosphorylates transformer 2β1 (Tra2β1) at numerous sites regulating RNA binding, splicing of the survival motor neuron 2 gene, and catalytic function of the kinase domain. Conclusion: The two RS domains interact and regulate Tra2β1 activity in a phosphorylation-dependent manner. Significance: SRPK1 is a regulator of Tra2β1.

Most annotated genomes show a large number of sense-antisense transcripts that can generate double-stranded RNAs. We describe a method to clone these dsRNAs from total RNA preparations.

Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.

The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid–binding proteins YB-1 and human Tra2-β1. Here, we show that hTra2-β1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-β1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-β1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis. (Cancer Res 2006; 66(9): 4774-80)