SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3 0 splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.(A) Distribution of cryptic 3 0 ss around the associated canonical 3 0 ss. Red and black histograms indicate 1,145 cryptic 3 0 ss more frequently used (q value < 0.05) in six mutant SF3B1 MDS patient samples and 186 cryptic 3 0 ss in nine WT SF3B1 samples, respectively. (B) Hierarchical clustering and heatmap analysis of the 627 cryptic 3 0 ss differentially used in six mutant versus nine WT SF3B1 samples (q value < 0.05 and cryptic 3 0 ss closer than 100 nt upstream of the associated canonical 3 0 ss). Each row represents one cryptic 3 0 ss and each column one MDS patient sample. Z scores in the matrix represent normalized percent-spliced-in values. The color bars above the heatmap indicate the SF3B1 mutations. (C) Volcano plot representation of genes associated with the 169 cryptic 3 0 ss differentially used in mutant versus WT SF3B1 samples (q value < 0.05, closer than 50 nt upstream of the associated canonical 3 0 ss and more than 15 supporting reads averaged over mutant SF3B1 samples). The horizontal axis shows the PSI difference between mutant and WT SF3B1 samples, and the vertical axis shows the significance. Genes selected for further experimental validation are highlighted in red.

No abstract

Some neurological disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), fragile X syndrome, Huntington's disease, myotonic dystrophy, and various ataxias, can be caused by expansions of short nucleic acid sequence repeats in specific genes. A possible disease mechanism involves the transcribed repeat RNA binding an RNAbinding protein (RBP), resulting in its sequestration and thus dysfunction. Polycomb repressive complex 2 (PRC2), the histone methyltransferase that deposits the H3K27me3 mark of epigenetically silenced chromatin, binds G-rich RNAs and has especially high affinity for G-quadruplex (G-Q) structures. Here, we find that PRC2 target genes are derepressed and the RNA binding subunit EZH2 largely insoluble in postmortem brain samples from ALS/FTD patients with C9ORF72 (C9) repeat expansions, leading to the hypothesis that the (G 4 C 2 ) n repeat RNA might be sequestering PRC2. Contrary to this expectation, we found that C9 repeat RNAs (n = 6 or 10) bind weakly to purified PRC2, and studies with the G-Q specific BG4 antibody and circular dichroism studies both indicated that these C9 RNAs have little propensity to form G-Qs in vitro. Several GC-rich triplet-repeat expansion RNAs also have low affinity for PRC2 and do not appreciably form G-Qs in vitro. The results are consistent with these sequences forming hairpin structures that outcompete G-Q folding when the repeat length is sufficiently large. We suggest that binding of PRC2 to these GC-rich RNAs is fundamentally weak but may be modulated in vivo by protein factors that affect secondary structure, such as helicases and other RBPs.

Kindlin‐2 is engaged in tumor progression. However, the mechanism accounting for Kindlin‐2 regulation in tumor cells remained largely unknown. Here, we report a regulatory loop between Kindlin‐2 and GLI1, an effector of Hedgehog signaling pathway. We show that Kindlin‐2 is transcriptionally downregulated via GLI1 occupancy on the Kindlin‐2 promoter. Adversely, we found that Kindlin‐2 promotes GLI1 expression through a mechanism involving GSK3β inactivation and is independent of Smoothened. Functionally, knockdown of Kindlin‐2 cooperates with cyclopamine, a Smoothened antagonist, to decrease the viability of prostate cancer cells. Taken together, targeting the Kindlin‐2–GLI1 feedback loop may facilitate the killing of prostate cancer cells.