Project description:Nuclear transcriptional factors are key regulators of stem cell identity and differentiation, however whether extra-nuclear factors are involved in cell fate decisions remains unclear. Here we report that centrosome-associated spliceosome repression drives the differentiation of immature neuroblasts into neurons. Using neuroblastoma cells undergoing asymmetric division (ACD) as a model, we demonstrate that spliceosome components assemble preferentially at the mother centriole and undergo dynamic relocation in response to retinoic acid (RA) or centrosome ablation. ACD cells require selective Ninein splicing variants to promote the efficient formation of centrosome-associated spliceosome condensates. RA-induced neuron differentiation activates ciliogenesis and alternative splicing patterns, including cytoplasmic intron-retained transcripts in spliceosome-related genes. While pharmacological ablation of cilia impedes RA-dependent neuronal differentiation, inhibition of centriole duplication enhances synapse formation, recapitulating the molecular and morphological features of neurons. Mechanistically, centrosome repression-mediated differentiation of ACD cells into neurons relocates spliceosome factors between the nucleus and centrosome, activating cytoplasmic intron retention and exon inclusion in genes essential for ciliogenesis and cerebral cortical development. Collectively, our findings establish a cellular model for programmed splicing control and a strategy to enhance cytoplasmic spliced genes critical for human brain development.
Project description:SF3B1 is essential for assembly of B-complex spliceosome and activation of the Bact complex and undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle. We have recently showed that the N-terminus of SF3B1 is phosphorylated during spliceosome activation by a little studied cyclin-dependent kinase 11 (CDK11) and remains hyperphosphorylated in catalytically active spliceosomes. However, little is known about the function of this N-terminal phosphorylation, about factors that “read” it, or about a spliceosome intermediate generated by CDK11 targeting. Here we demonstrate that CDK11 inhibition blocks spliceosome in a new intermediate during B to Bact transition. We now show that, phosphorylated SF3B1 N-terminus is crucial for binding of proteins during spliceosome activation.
Project description:SF3B1 is essential for assembly of B-complex spliceosome and activation of the Bact complex and undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle. We have recently showed that the N-terminus of SF3B1 is phosphorylated during spliceosome activation by a little studied cyclin-dependent kinase 11 (CDK11) and remains hyperphosphorylated in catalytically active spliceosomes. However, little is known about the function of this N-terminal phosphorylation, about factors that “read” it, or about a spliceosome intermediate generated by CDK11 targeting. Here we demonstrate that CDK11 inhibition blocks spliceosome in a new intermediate during B to Bact transition. We now show that, phosphorylated SF3B1 N-terminus is crucial for binding of proteins during spliceosome activation.
Project description:The studies of spliceosomal interactions are challenging due to their dynamic nature. Here we developed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs) to simultaneously map the spliceosomal binding to human snRNAs and pre-mRNAs. This identified 9 distinct regions on pre-mRNAs, which overlap with position-dependent binding patterns of 15 RBPs. Using spliceosome iCLIP, we additionally identified >50,000 branchpoints (BPs) that have canonical features, unlike those identified by RNA-seq. The iCLIP BPs generally overlap with the computationally predicted BPs, and alternative BPs are associated with extended regions of structurally accessible RNA. We find that the position and strength of BPs defines the binding patterns of SF3 and U2AF complexes, whereas the RNA structure around BPs affects the sensitivity of exons to perturbation of these complexes. Our findings introduce spliceosome iCLIP as a new method for transcriptomic studies of BPs and splicing mechanisms.
Project description:microRNA dysregulation is a common feature of cancer cells, but the complex roles of microRNAs in cancer are not fully elucidated. Here we used functional genomics to identify oncogenic microRNAs in non-small cell lung cancer and to evaluate their impact on response to EGFR targeting therapy. Our data demonstrate that microRNAs with an AAGUGC-motif in their seed-sequence increase both cancer cell proliferation and sensitivity to EGFR inhibitors. Global transcriptomics, proteomics and target prediction resulted in the identification of several tumor suppressors involved in the G1/S transition as targets of AAGUGC-microRNAs. The clinical implications of our findings were evaluated by analysis of public domain data supporting the link between this microRNA seed-family, their tumor suppressor targets and cancer cell proliferation. In conclusion we propose that AAGUGC-microRNAs are an integral part of an oncogenic signaling network, and that these findings have potential therapeutic implications, especially in selecting patients for EGFR-targeting therapy.
Project description:Alternative splicing (AS) can produce multiple transcripts with different exon-intron structures from a single pre-mRNA. Pre-mRNA splicing is catalyzed by a dynamic macromolecular ribonucleoprotein (RNP) complex termed the spliceosome. The spliceosome consists of several small nuclear ribonucleoproteins (snRNPs) that bind uridine-rich small nuclear RNA (snRNA). In U1, U2, U4 and U5 snRNPs, snRNA interacts with the conserved Smith antigen (Sm) proteins via a bipartite Sm sequence motif. In eukaryotes, seven Sm proteins (B, D1/2/3, E, F and G) form a heptameric ring-shaped complex surrounding the snRNA. Here, we performed a tandem affinity purification using SMEB as a bait in Arabidopsis cell suspension cultures. At least 45 known/hypothesized and potential novel spliceosome components were identified in Arabidopsis.