Evolution of the U2 Spliceosome for Processing Numerous and Highly Diverse Non-canonical Introns in the Chordate Fritillaria borealis.

An overwhelming majority of eukaryotic introns have GT/AG ends, whose identities play a critical role for their recognition and removal by the U2 spliceosome, a well-conserved complex of protein and RNAs. Introns with other splice sites exist at very low frequencies in various genomes, and some of them are processed by the U12 spliceosome. Here, we show that, in the chordate Fritillaria borealis, the majority of old introns have been lost and replaced by introns with highly divergent splice sites. The new introns of F. borealis are exceptionally diverse, though more frequently AG/AC or AG/AT, and features of thousands of them support an origin from transposons. They cannot be processed in human cells, but their splicing is rescued by mutating terminal dinucleotides to GT/AG. With lariat sequencing and splicing inhibitor assays, we show that F. borealis introns are spliced by the U2 spliceosome, which thus evolved to a different selectivity, with neither novel U1 small nuclear RNA (snRNA) types nor major remodeling of its protein and snRNA complements. This genome-wide recolonization by non-canonical introns emphasizes the importance of transposons as a resource of novel introns in a context of massive intron loss. An evolution of the spliceosome may also permit to neutralize harmful transposons through their conversion into introns.

Notch1 signaling in NOTCH1-mutated mantle cell lymphoma depends on Delta-Like ligand 4 and is a potential target for specific antibody therapy.

BACKGROUND: NOTCH1 gene mutations in mantle cell lymphoma (MCL) have been described in about 5-10% of cases and are associated with significantly shorter survival rates. The present study aimed to investigate the biological impact of this mutation in MCL and its potential as a therapeutic target. METHODS: Activation of Notch1 signaling upon ligand-stimulation and inhibitory effects of the monoclonal anti-Notch1 antibody OMP-52M51 in NOTCH1-mutated and -unmutated MCL cells were assessed by Western Blot and gene expression profiling. Effects of OMP-52M51 treatment on tumor cell migration and tumor angiogenesis were evaluated with chemotaxis and HUVEC tube formation assays. The expression of Delta-like ligand 4 (DLL4) in MCL lymph nodes was analyzed by immunofluorescence staining and confocal microscopy. A MCL mouse model was used to assess the activity of OMP-52M51 in vivo. RESULTS: Notch1 expression can be effectively stimulated in NOTCH1-mutated Mino cells by DLL4, whereas in the NOTCH1-unmutated cell line JeKo-1, less effect was observed upon any ligand-stimulation. DLL4 was expressed by histiocytes in both, NOTCH1-mutated and -unmutated MCL lymph nodes. Treatment of NOTCH1-mutated MCL cells with the monoclonal anti-Notch1 antibody OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed the induction of numerous direct Notch target genes involved in lymphoid biology, lymphomagenesis and disease progression. Importantly, in lymph nodes from primary MCL cases with NOTCH1/2 mutations, we detected an upregulation of the same gene sets as observed in DLL4-stimulated Mino cells. Furthermore, DLL4 stimulation of NOTCH1-mutated Mino cells enhanced tumor cell migration and angiogenesis, which could be abolished by treatment with OMP-52M51. Importantly, the effects observed were specific for NOTCH1-mutated cells as they did not occur in the NOTCH1-wt cell line JeKo-1. Finally, we confirmed the potential activity of OMP-52M51 to inhibit DLL4-induced Notch1-Signaling in vivo in a xenograft mouse model of MCL. CONCLUSION: DLL4 effectively stimulates Notch1 signaling in NOTCH1-mutated MCL and is expressed by the microenvironment in MCL lymph nodes. Our results indicate that specific inhibition of the Notch1-ligand-receptor interaction might provide a therapeutic alternative for a subset of MCL patients.