Exploration of the effects of the CYCLOPS gene RBM17 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal and malignant tumours worldwide. New therapeutic targets for HCC are urgently needed. CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes have been noted to be associated with cancer-targeted therapies. Therefore, we intended to explore the effects of the CYCLOPS gene RBM17 on HCC oncogenesis to determine if it could be further used for targeted therapy. METHODS: We collected data on 12 types of cancer from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) queries for comparison with adjacent non-tumour tissues. RBM17 expression levels, clinicopathological factors and survival times were analysed. RNAseq data were downloaded from the Encyclopaedia of DNA Elements database for molecular mechanism exploration. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when RBM17 expression was inhibited by shRBM17. Cell cycle progression and apoptosis were also examined to investigate the pathogenesis of RBM17. RESULTS: Based on 6,136 clinical samples, RBM17 was markedly overexpressed in most cancers, especially HCC. Moreover, data from 442 patients revealed that high RBM17 expression levels were related to a worse prognosis. Overexpression of RBM17 was related to the iCluster1 molecular subgroup, TNM stage, and histologic grade. Pathway analysis of RNAseq data suggested that RBM17 was involved in mitosis. Further investigation revealed that the proliferation rates of HepG2 (P = 0.003) and SMMC-7721 (P = 0.030) cells were significantly reduced when RBM17 was knocked down. In addition, RBM17 knockdown also arrested the progression of the cell cycle, causing cells to halt at the G2/M phase. Increased apoptosis rates were also found in vitro. CONCLUSION: These results suggest that RBM17 is a potential therapeutic target for HCC treatment.

Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo.

Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this 'feed-back' effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.

Neural crest-specific deletion of Rbfox2 in mice leads to craniofacial abnormalities including cleft palate.

Alternative splicing (AS) creates proteomic diversity from a limited size genome by generating numerous transcripts from a single protein-coding gene. Tissue-specific regulators of AS are essential components of the gene regulatory network, required for normal cellular function, tissue patterning, and embryonic development. However, their cell-autonomous function in neural crest development has not been explored. Here, we demonstrate that splicing factor Rbfox2 is expressed in the neural crest cells (NCCs), and deletion of Rbfox2 in NCCs leads to cleft palate and defects in craniofacial bone development. RNA-Seq analysis revealed that Rbfox2 regulates splicing and expression of numerous genes essential for neural crest/craniofacial development. We demonstrate that Rbfox2-TGF-ß-Tak1 signaling axis is deregulated by Rbfox2 deletion. Furthermore, restoration of TGF-ß signaling by Tak1 overexpression can rescue the proliferation defect seen in Rbfox2 mutants. We also identified a positive feedback loop in which TGF-ß signaling promotes expression of Rbfox2 in NCCs.

PUF60-activated exons uncover altered 3' splice-site selection by germline missense mutations in a single RRM.

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development.

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3' UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3' UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3' end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.

Rbfox1 Regulates Synaptic Transmission through the Inhibitory Neuron-Specific vSNARE Vamp1.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

Physiologic Expression of Sf3b1(K700E) Causes Impaired Erythropoiesis, Aberrant Splicing, and Sensitivity to Therapeutic Spliceosome Modulation.

More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.

RBFox2 Binds Nascent RNA to Globally Regulate Polycomb Complex 2 Targeting in Mammalian Genomes.

Increasing evidence suggests that diverse RNA binding proteins (RBPs) interact with regulatory RNAs to regulate transcription. RBFox2 is a well-characterized pre-mRNA splicing regulator, but we now encounter an unexpected paradigm where depletion of this RBP induces widespread increase in nascent RNA production in diverse cell types. Chromatin immunoprecipitation sequencing (ChIP-seq) reveals extensive interaction of RBFox2 with chromatin in a nascent RNA-dependent manner. Bayesian network analysis connects RBFox2 to Polycomb complex 2 (PRC2) and H3K27me3, and biochemical experiments demonstrate the ability of RBFox2 to directly interact with PRC2. Strikingly, RBFox2 inactivation eradicates PRC2 targeting on the majority of bivalent gene promoters and leads to transcriptional de-repression. Together, these findings uncover a mechanism underlying the enigmatic association of PRC2 with numerous active genes, highlight the importance of gene body sequences to gauge transcriptional output, and suggest nascent RNAs as critical signals for transcriptional feedback control to maintain homeostatic gene expression in mammalian genomes.