Investigating the mechanism of alternative splicing regulation of the RNA-binding proteins T-STAR and Sam68

PhD ThesisAlternative splicing is an important mechanism of pre-mRNA processing, regulated by many splicing factors. Some splicing factors as T-STAR, are poorly characterised due to absence of RNA targets identified. The first main aim of this study was to identify the principles for splicing regulation by T-STAR, by characterising its first identified physiological targets Neurexin-2 and Tomosyn-2. The second important aim was to compare the T-STAR and Sam68 splicing regulation on example of Neurexin-2 being a T-STAR specific target, and Tomosyn-2 being regulated by both T-STAR and Sam68. By EMSA analyses, I identified that Neurexin-2 is a direct target of both T-STAR and Sam68. By minigene assays and mutagenesis, I found that T-STAR response element in Neurexin-2 is composite. Individually each AU-rich region is redundant for splicing regulation, but loss of two key groups of AU-rich sequence elements inhibits splicing control by T-STAR. Several splicing regulator proteins have been shown to follow a pattern where binding upstream of a regulated exon leads to splicing repression and binding downstream leads to splicing activation. The T-STAR response element starts just 13 nucleotides downstream of the regulated Neurexin-2 exon, suggesting T-STAR protein binding might physically occlude the 5´splice site. Instead, I found this T-STAR response element can still potently repress splicing even from more distant downstream locations, and surprisingly even when placed upstream of the regulated exon. To find out how general this form of position-independent splicing regulation is I identified AU-rich downstream sequence elements that control splicing patterns of Tomosyn-2 in response to both T-STAR and Sam68. These Tomosyn-2 splicing response sequences similarly repressed splicing when moved upstream of the regulated exon. The T-STAR response element in Neurexin-2 predominantly contained UUAA sequences, while the T-STAR/Sam68 response element in Tomosyn-2 contained UAAA repeats. Conversion of the Neurexin response element to UAAA and UAAAA placed this exon under control of both Sam68 and T-STAR. Current data suggest that T-STAR and Sam68 proteins are unusual in that they repress splicing from either side of the exon. These proteins have subtly different target sequences that can enable them to control distinct patterns of target exons in the cell

Inter and intra-tumoral heterogeneity as a platform for personalized therapies in medulloblastoma

Medulloblastoma is the most common malignant CNS tumor of childhood, affecting ~350 patients/year in the USA. In 2020, most children are cured of their disease, however, survivors are left with life-long late-effects as a consequence of intensive surgery, and application of chemo- and radio-therapy to the developing brain. The major contributor to improvements in patient survival has been the development of risk-stratified treatments derived from a better understanding of the prognostic value of disease biomarkers. The characterization and validation of these biomarkers has engendered a comprehensive understanding of the extensive heterogeneity that exists within the disease, which can occur both between and within tumors (inter- and intra-tumoral heterogeneity, respectively). In this review, we discuss inter-tumoral heterogeneity, describing the early characterization of clinical and histopathological disease heterogeneity, the more recent elucidation of molecular disease subgroups, and the potential for novel therapies based on specific molecular defects. We reflect on the limitations of current approaches when applied to a rare disease. We then review early investigations of intra-tumoral heterogeneity using FISH and immunohistochemical approaches, and focus on the application of next generation sequencing on bulk tumors to elucidate intra-tumoral heterogeneity. Finally, we critically appraise the applications of single-cell sequencing approaches and discuss their potential to drive next biological insights, and for routine clinical application

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons

Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α–Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome

Spectrum of Genetic Changes in Patients with Non-Syndromic Hearing Impairment and Extremely High Carrier Frequency of 35delG GJB2 Mutation in Belarus

The genetic nature of sensorineural hearing loss (SNHL) has so far been studied for many ethnic groups in various parts of the world. The single-nucleotide guanine deletion (35delG) of the GJB2 gene coding for connexin 26 was shown to be the main genetic cause of autosomal recessive deafness among Europeans. Here we present the results of the first study of GJB2 and three mitochondrial mutations among two groups of Belarusian inhabitants: native people with normal hearing (757 persons) and 391 young patients with non-syndromic SNHL. We have found an extremely high carrier frequency of 35delG GJB2 mutation in Belarus −5.7%. This point deletion has also been detected in 53% of the patients with SNHL. The 312del14 GJB2 was the second most common mutation in the Belarus patient cohort. Mitochondrial A1555G mt-RNR1 substitution was found in two SNHL patients (0.55%) but none were found in the population cohort. No individuals carried the A7445G mutation of mitochondrial mt-TS1. G7444A as well as T961G substitutions were detected in mitochondrial mt-RNR1 at a rate of about 1% both in the patient and population cohorts. A possible reason for Belarusians having the highest mutation carrier frequency in Europe 35delG is discussed

Transcriptomic profiling of human skin biopsies in the clinical trial setting: A protocol for high quality RNA extraction from skin tumours [version 1; referees: 2 approved, 1 approved with reservations]

Transcriptomic profiling of skin disease using next generation sequencing allows for detailed information on aspects of RNA biology including gene expression, non-coding regulatory elements and gene splicing. The application of RNA sequencing to human skin disease and cancer is often hampered by degraded RNA. Here we describe a protocol that allows for consistently intact RNA to be extracted from snap frozen skin biopsy samples, which has been validated in a clinical trial setting. Human skin tumour punch biopsies (n=28) ranging from 4-6mm in diameter were obtained from 14 patients with an inherited skin tumour syndrome (CYLD cutaneous syndrome) and frozen in liquid nitrogen prior to being stored at -80°C. These samples were then subject to cyrostat sectioning, allowing for histological assessment, and were homogenised using a bead-based lysis platform. RNA extraction was performed using a silica column-based system. RNA concentration was measured using fluorescent quantitation and RNA integrity assessed using microfluidic gel electrophoresis. We also processed normal skin biopsies using the same protocol (n=10). The mean RNA integrity score of the tumour and normal samples was 9.5, and the quantity of RNA obtained from the small amounts of tissue used exceeded requirements for RNA-seq library generation. We propose that the method of RNA extraction suggested here allows for transcriptomic profiling from small pieces of human tissue without the need for PCR amplification during library preparation. This protocol could be utilised in healthy and diseased skin to improve mechanistic understanding in a range of human skin diseases

Curcumin and Carnosic Acid Cooperate to Inhibit Proliferation and Alter Mitochondrial Function of Metastatic Prostate Cancer Cells

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality