Characterization of the regulation of CD46 alternative splicing

Alternative splicing gives rise to multiple mRNA and protein isoforms. This process is catalyzed by the spliceosome and regulated by cis-acting elements, trans-acting factors, transcription and chromatin structure. CD46 is a membrane-bound complement control protein with several extracellular STP domain variants and two mutually exclusive cytoplasmic tails derived from the alternative splicing of cassette exons 7, 8 and 9, or cassette exon 13, respectively. The two cytoplasmic tails have different signaling capacities and regulatory functions in T helper 1 cells and epithelial cells. Besides, the association between aberrant CD46 alternative splicing and autoimmune diseases proposes a possible novel therapy by amending splicing. To understand the splicing regulatory mechanisms of CD46, serial deletion assays in splicing minigenes were performed to identify cis-acting elements while the loss- and gain-of-function assays as well as RNA pull-down were carried out to determine the roles of protein regulators. SRSF1 and PTBP1 repress exon 13 inclusion via exonic splicing silencers. SRSF1 is a common activator yet it inhibits exon 13 inclusion in this context and it appears to do so by direct binding within this exon. On the other hand, the splicing activators, TIA1 and TIAL1, strongly enhance exon 13 recognition via the poly-U rich sequences downstream of the 5'ss. The alternative splicing pattern of exon 13 was efficiently modulated by anti-sense oligonucleotides targeting splicing enhancers or silencers in a dose-dependent manner but not by T-cell signaling. From these studies, a better understanding for CD46 alternative splicing regulation was achieved yet much remains to be elucidated to fully decipher the CD46 alternative splicing mechanisms. In conclusion, splicing regulation is complicated and context-dependent, and thus intensive studies of alternative splicing events are necessary to improve the current knowledge. DOCTOR OF PHILOSOPHY (SBS

Alternative polyadenylation expands the mRNA isoform repertoire of human CD46

Alternative polyadenylation is a prevalent mechanism regulating mammalian gene expression. While tandem 3′-Untranslated-Region (3′UTR) polyadenylation changes expression levels, Intronic PolyAdenylation generates shorter transcripts encoding truncated proteins. Intronic PolyAdenylation regulates 20% of genes and is especially common in receptor tyrosine-kinase transcripts, generating soluble repressors. Here we report that human CD46, encoding a TransMembrane repressor of complement and T-cell co-stimulator, expresses multiple isoforms by alternative polyadenylation. We provide evidence for polyadenylation at several introns by RT-PCR of 5′ intronic fragments, and by increase in such isoforms via functional U1 knockdown. We mapped various Intronic PolyAdenylation Sites by 3′ Rapid Amplification of cDNA Ends (3′RACE), which could generate soluble or membrane-bound but tail-less CD46. Intronic PolyAdenylation could add to the source of soluble CD46 isoforms in fluids and tissues, which increase in cancers and autoimmune syndromes. Furthermore, 3′RACE identified three PolyAdenylation Sites within the last intron and exon, whose transcripts with shortened 3′UTRs could support higher CD46 expression. Finally, 3′RACE revealed that the CD46 Pseudogene only expresses short transcripts by early polyadenylation in intron 2. Overall, we report a wide variety of CD46 mRNA isoforms which could generate new protein isoforms, adding to the diverse physiological and pathological roles of CD46.MOE (Min. of Education, S’pore)Accepted versio

Characterization of the regulation of CD46 RNA alternative splicing

Here we present a detailed analysis of the alternative splicing regulation of human CD46, which generates different isoforms with distinct functions. CD46 is a ubiquitous membrane protein that protects host cells from complement and plays other roles in immunity, autophagy, and cell adhesion. CD46 deficiency causes an autoimmune disorder, and this protein is also involved in pathogen infection and cancer. Before this study, the mechanisms of CD46 alternative splicing remained unexplored even though dysregulation of this process has been associated with autoimmune diseases. We proved that the 5′ splice sites of CD46 cassette exons 7 and 8 encoding extracellular domains are defined by noncanonical mechanisms of base pairing to U1 small nuclear RNA. Next we characterized the regulation of CD46 cassette exon 13, whose inclusion or skipping generates different cytoplasmic tails with distinct functions. Using splicing minigenes, we identified multiple exonic and intronic splicing enhancers and silencers that regulate exon 13 inclusion via trans-acting splicing factors like PTBP1 and TIAL1. Interestingly, a common splicing activator such as SRSF1 appears to repress CD46 exon 13 inclusion. We also report that expression of CD46 mRNA isoforms is further regulated by non-sense-mediated mRNA decay and transcription speed. Finally, we successfully manipulated CD46 exon 13 inclusion using antisense oligonucleotides, opening up opportunities for functional studies of the isoforms as well as for therapeutics for autoimmune diseases. This study provides insight into CD46 alternative splicing regulation with implications for its function in the immune system and for genetic disease.MOE (Min. of Education, S’pore)Published versio

Correlations between Percolation Threshold, Dispersion State, and Aspect Ratio of Carbon Nanotubes

Critical factors that determine the percolation threshold of carbon nanotube (CNT)-reinforced polymer nanocomposites are studied. An improved analytical model is developed based on an interparticle distance concept. Two dispersion parameters are introduced in the model to correctly reflect the different dispersion states of CNTs in the matrix - entangled bundles and well-dispersed individual CNTs. CNT-epoxy nanocomposites with different dispersion states are fabricated from the same constituent materials by employing different processing conditions. The corresponding percolation thresholds of the nanocomposites vary over a wide range, from 0.1 to greater than 1.0 wt \%, and these variations are explained in terms of dispersion parameters and aspect ratios of CNTs. Important factors that control the percolation threshold of nanocomposites are identified based on the comparison between modeling data and experimental results

Cis- and trans-regulations of pre-mRNA splicing by RNA editing enzymes influence cancer development

10.1038/s41467-020-14621-5NATURE COMMUNICATIONS11

Bidirectional regulation of adenosine-to-inosine (A-to-I) RNA editing by DEAH box helicase 9 (DHX9) in cancer

10.1093/nar/gky396NUCLEIC ACIDS RESEARCH46157953-796