Target pre-mRNA Research Articles

Characterization of cis-acting elements that control oscillating alternative splicing

ABSTRACTAlternative splicing (AS) in response to changing external conditions often requires alterations in the ability of sequence-specific RNA-binding proteins to bind to cis-acting sequences in their target pre-mRNA. While daily oscillations in AS events have been described in several organisms, cis-acting sequences that control time of the day-dependent AS remain largely elusive. Here we define cis-regulatory RNA elements that control body-temperature driven rhythmic AS using the mouse U2af26 gene as a model system. We identify a complex network of cis-regulatory sequences that regulate AS of U2af26, and show that the activity of two enhancer elements is necessary for oscillating AS. A minigene comprising these U2af26 regions recapitulates rhythmic splicing of the endogenous gene, which is controlled through temperature-regulated SR protein phosphorylation. Mutagenesis of the minigene delineates the cis-acting enhancer element for SRSF2 within exon 6 to single nucleotide resolution and reveals that the combined activity of SRSF2 and SRSF7 is required for oscillating U2af26 AS. By combining RNA-Seq with an siRNA screen and individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP), we identify a complex network of SR proteins that globally controls temperature-dependent rhythmic AS, with the direction of splicing depending on the position of the cis-acting elements. Together, we provide detailed insights into the sequence requirements that allow trans-acting factors to generate daily rhythms in AS.

Abstract LB-323: A novel function of ETS transcription factors in alternative splicing is altered in oncogenic FET-ETS fusions

Abstract Ewing sarcomas are characterized by fusions proteins between FET (FUS, EWS and TAF15) family genes encoding RNA binding proteins and ETS (ERG, FLI1 and FEV) family genes encoding transcription factors. FET-ETS fusions regulate both transcription and alternative splicing programs in Ewing sarcoma cells. To date, the function of FET-ETS fusions in splicing is mostly attributed to the FET N-terminal region which is preserved in the fusions. We recently demonstrated that wild type ERG associates with poly-adenylated mRNA and control mRNAs stability in the cytoplasm (Rambout et al., 2016). This therefore indicates that ERG proteins might not be considered only as transcription factors, but can also regulate post-transcriptional processes. On this ground, we hypothesize that ERG proteins might control other post-transcriptional processes, including splicing. In this study, we show that the ERG transcription factor interacts with several spliceosome components and is associated with nascent RNA. Tethered ERG proteins controlled alternative splicing of a minigene reporter in an orientation-specific manner. Deep RNA-seq identified hundreds of alternatively spliced exons regulated by ERG depletion, with an enrichment in RNA motifs bound by RBFOX2 splicing factor. RBFOX2 similarly regulated alternative splicing of common targets and interacted together via the C-terminal domain of ERG which is present in FET-ERG fusions. In Ewing sarcoma cells, RNA-seq identified hundreds of alternatively spliced exons regulated by EWS-FLI1 depletion, with an enrichment in RNA motifs bound by RBFOX2. The oncogenic EWS-FLI1 protein binds RBFOX2 protein but has an antagonist effect on RBFOX2 function. Cross-linking immunoprecipitation (CLIP) experiments show that the EWS-FLI1 fusion inhibits RBFOX2 function by repressing its binding to target pre-mRNAs. We hypothesize that the low complexity prion-like domain of EWS included in the fusion protein may be responsible for sequestration of RBFOX2 outside of the splicing machinery. Importantly, we also show that some of the splicing events induced by the fusion protein may play a critical role in FET-ETS induced cell phenotype. Specific splicing switch partially recapitulate the phenotypic changes that participate in FET-ETS induced cell transformation. These data reveal a new role for ERG family proteins in alternative splicing that is altered in FET-ETS oncogenic proteins, thereby contributing to alternative splicing mis-regulation and cell phenotype in Ewing sarcoma. Citation Format: Olivier Saulnier, Katia Guerdi, Marie-Ming Aynaud, Alina Chakraborty, Josephine Pineau, Christina O Grady, Martin Dutertre, Franck Dequiedt, Olivier Delattre. A novel function of ETS transcription factors in alternative splicing is altered in oncogenic FET-ETS fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-323.

Cancer-type organic anion transporting polypeptide 1B3 is a target for cancer suicide gene therapy using RNA trans-splicing technology

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) has been identified as a cancer-specific transcript in various solid cancers, including colorectal cancer. Given its excellent cancer-specific expression profile, we hypothesized that Ct-OATP1B3 could represent a promising target for cancer-specific expression of the suicide gene, herpes simplex virus 1 thymidine kinase (HSV-tk), via a spliceosome-mediated RNA trans-splicing (SMaRT) approach. SMaRT technology is used to recombine two RNA molecules to generate a chimeric transcript. In this study, we engineered an RNA trans-splicing molecule carrying a translation-defective HSV-tk sequence (RTM44), which was capable of inducing its own trans-splicing to the desired Ct-OATP1B3 pre-mRNA target. RTM44 expression in LS180 cells resulted in generation of Ct-OATP1B3/HSV-tk fusion mRNA. A functional translation start site contributed by the target pre-mRNA restored HSV-tk protein expression, rendering LS180 cells sensitive to ganciclovir treatment in vitro and in xenografted mice. The observed effects are ascribed to accurate and efficient trans-splicing, as they were absent in cells carrying a splicing-deficient mutant of RTM44. Collectively, our data highlights Ct-OATP1B3 as an ideal target for the HSV-tk SMaRT suicide system, which opens up new translational avenues for Ct-OATP1B3-targeted cancer therapy.

FOXP3 interacts with hnRNPF to modulate pre-mRNA alternative splicing

FOXP3 promotes the development and function of regulatory T cells mainly through regulating the transcription of target genes. RNA alternative splicing has been implicated in a wide range of physiological and pathophysiological processes. We report here that FOXP3 associates with heterogeneous nuclear ribonucleoprotein (hnRNP) F through the exon 2-encoded region of FOXP3 and the second quasi-RNA recognition motif (qRRM) of hnRNPF. FOXP3 represses the ability of hnRNPF to bind to its target pre-mRNA and thus modulates RNA alternative splicing. Furthermore, overexpression of mouse hnRNPF in in vitro-differentiated regulatory T cells (Tregs) reduced their suppressive function. Thus, our studies identify a novel mechanism by which FOXP3 regulates mRNA alternative splicing to modulate the function of regulatory T cells.

Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA

BackgroundRBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform.ResultsRBM10 knockdown (KD) provoked alterations in splicing events in 10–20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity.ConclusionOur work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

167 Long-term in vivo correction of a recessive dystrophic epidermolysis bullosa phenotype using RNA trans-splicing repair

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

169 Regeneration of a functional epidermis at a large, long-standing wound by gene-corrected autologous epidermal stem cells

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3' UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG)n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG)n-MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG)n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the β-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases.

(1)H, (15)N and (13)C backbone and side chain resonance assignments of the RRM domain from human RBM24.

Tissue development requires the expression of a regulated subset of genes, and it is becoming clear that the process of alternative splicing also plays an important role in the production of necessary tissue-specific isoforms. However, only a few of these tissue-specific splicing factors in mammals have so far been discovered. One of these factors is the RNA-binding protein RBM24 which has been recently identified as a major regulator of alternative splicing in cardiac and skeletal muscle development. The RBM24 protein contains an RNA recognition motif (RRM) domain that presumably mediates the binding to target pre-mRNA required for regulation of the splicing patterns. Here we report (1)H, (15)N and (13)C chemical shift assignments of the backbone and sidechain atoms for the RRM domain from human RBM24. Secondary chemical shift analysis and relaxation measurement confirm the canonical architecture of the RRM domain. The data will allow for atomic level studies aimed at understanding splicing regulation of target genes in heart and muscle development and investigation into a separate role of RBM24 in modulating mRNA stability of genes involved in the p53 tumor suppressor pathway.

Abstract B18: Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent

Abstract African American (AA) men exhibit a nearly 2-fold higher incidence and 3-fold higher mortality rate from prostate cancer compared to Caucasian American (CA) men and disparities in tumor aggressiveness remain after controlling for social determinants of health. Previous work from our laboratory and others has revealed differences in gene expression that contribute to prostate cancer health disparities among AAs. Oncogenic signaling pathways exhibiting up-regulation more often in AA prostate cancer include androgen receptor (AR) and epidermal growth factor receptor (EGFR). Thus, novel therapeutic strategies capable of driving production of inhibitory AR and EGFR isoforms and capable of limiting aberrant constitutively active AR isoforms are urgently needed. Such strategies will increase our understanding of these molecular mechanisms underlying prostate cancer in AA men. In addition, such strategies have the potential to ultimately result in novel specific approaches for treatment that will help reduce prostate cancer disparities for AAs and will improve treatment of advanced stage disease in all men with aggressive disease driven by these mechanisms. Splice-switching oligonucleotides (SSOs) represent a novel therapeutic strategy to combat prostate cancer. Unlike the strategy of using RNA interference to inhibit the expression of a gene, SSOs simultaneously limit the production of pathogenic proteins and induce the expression of protein variants with therapeutic value. SSOs modulate pre-mRNA splicing by binding to target pre-mRNAs and blocking access of the splicing machinery to a particular splice site, and can be used to produce novel splice variants or correct aberrant splicing. Herein we have designed and synthesized novel SSOs targeting AR and EGFR pre-mRNAs to produce inhibitory splice variants and correct aberrant splicing as follows. We have synthesized a SSO to correct the aberrant splicing that leads to production of the constitutively active AR-V7 variant leading to restoration of ligand dependency. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the cryptic exon included in AR-V7, decreases AR-V7 RNA. In addition, we have synthesized a SSO to drive production of a naturally occurring dominant-negative AR variant (AR45) leading to interference with wild type AR transactivation activity. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the first exon of wild type AR and leads to inclusion of an alternative first exon encoding the unique N-terminal extension characterizing AR45, increases AR45 RNA. Furthermore, we have synthesized SSOs targeting exons in the transmembrane domain and the tyrosine kinase domain of EGFR to drive production of a soluble, naturally expressed inhibitory EGFR isoform and a dominant-negative EGFR isoform, respectively. Transfection of prostate cancer cells with these chemically modified SSOs increase RNA corresponding to these isoforms. Studies are underway to examine the effects of these SSOs on AR and EGFR signaling, respectively, and the effects of these SSOs on prostate tumor cell biology. These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs have the potential to further our understanding of the contribution of the targeted molecular mechanisms to AA prostate cancer and have the potential to yield novel therapeutic modalities to combat prostate cancer in AA men as well as men of all races with aggressive disease driven by these mechanisms. Citation Format: Jennifer A. Freedman, Timothy J. Robinson, Bonnie LaCroix, Brendon M. Patierno, Daniel J. George, Bruce A. Sullenger, Steven R. Patierno. Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B18.

CstF-64 and 3'-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα.

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.

RNA-binding protein CELF1 promotes tumor growth and alters gene expression in oral squamous cell carcinoma.

The RNA binding protein CELF1 (also known as CUGBP1) is emerging as a critical regulator of cancer cell proliferation and apoptosis. Here, to provide a global prospective of CELF1 regulation of oral squamous cell carcinoma, we performed RNA-sequencing in oral cancer cells and CELF1 overexpression analysis in non-malignant human oral keratinocytes. Our approaches identified 1283 mRNAs differentially regulated as a function of CELF1 expression and more importantly CELF1 promoted alternative splicing of several target pre-mRNAs, which are known to be involved in various cancer biological processes. Overexpression of CELF1 in non-malignant human oral keratinocytes protected cells against oxidative damage and altered gene expression patterns. Finally, we provide evidence that reduction of CELF1 protein using a xenograft tumorigenesis mouse model decreased tumor growth. Altogether, these data provided a comprehensive view of the CELF1 mRNA regulatory network in oral cancer and suggests that CELF1 and/or its target mRNAs are viable candidates for therapeutic intervention.

XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides

Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5′ to 3′ exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3′ fragments of the target RNAs in vitro. We found that the 3′ fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5′ ends by nuclear XRN2 after RNase H1-mediated cleavage, whereas the 3′ fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3′ fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA.

The fission yeast MTREC complex targets CUTs and unspliced pre-mRNAs to the nuclear exosome.

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome. However, the mechanism by which they are recognized and targeted to the exosome is not fully understood. Here we report that the MTREC complex, which has recently been shown to promote degradation of meiotic mRNAs and regulatory ncRNAs, is also the major nuclear exosome targeting complex for CUTs and unspliced pre-mRNAs in Schizosaccharomyces pombe. The MTREC complex specifically binds to CUTs, meiotic mRNAs and unspliced pre-mRNA transcripts and targets these RNAs for degradation by the nuclear exosome, while the TRAMP complex has only a minor role in this process. The MTREC complex physically interacts with the nuclear exosome and with various RNA-binding and RNA-processing complexes, coupling RNA processing to the RNA degradation machinery. Our study reveals the central role of the evolutionarily conserved MTREC complex in RNA quality control, and in the recognition and elimination of CUTs.

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies.

SF3B1 association with chromatin determines splicing outcomes.

Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. One probable explanation lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicate that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses reveal that SF3B1 specifically binds nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affects splicing of these exons similarly to SF3B1 knockdown. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice-site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure.

Synthesis and tau RNA binding evaluation of ametantrone-containing ligands.

We describe the synthesis and characterization of ametantrone-containing RNA ligands based on the derivatization of this intercalator with two neamine moieties (Amt-Nea,Nea) or with one azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as its combination with guanidinoneamine (Amt-NeaG4). Biophysical studies revealed that guanidinylation of the parent ligand (Amt-Nea) had a positive effect on the binding of the resulting compound for Tau pre-mRNA target as well as on the stabilization upon complexation of some of the mutated RNA sequences associated with the development of tauopathies. Further studies by NMR revealed the existence of a preferred binding site in the stem-loop structure, in which ametantrone intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4. Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity for disease-causing RNA molecules.