The Arabidopsis spliceosomal protein SmEb modulates ABA responses by maintaining proper alternative splicing of HAB1

Abscisic acid (ABA) signaling is critical for seed germination and abiotic stress responses in terrestrial plants. Pre-mRNA splicing is known to regulate ABA signaling. However, the involvement of canonical spliceosomal components in regulating ABA signaling is poorly understood. Here, we show that the spliceosome component Sm core protein SmEb plays an important role in ABA signaling. SmEb expression is up-regulated by ABA treatment, and analysis of Arabidopsis smeb mutant plants suggest that SmEb modulates the alternative splicing of the ABA signaling component HAB1 by enhancing the HAB1.1 splicing variant while repressing HAB1.2. Overexpression of HAB1.1 but not HAB1.2 rescues the ABA-hypersensitive phenotype of smeb mutants. Mutations in the transcription factor ABI3, 4, or 5 also reduce the ABA hypersensitivity of smeb mutants during seed germination. Our results show that the spliceosomal component SmEb plays an important role in ABA regulation of seed germination and early seedling development.

CLK1 reorganizes the splicing factor U1-70K for early spliceosomal protein assembly

Early spliceosome assembly requires phosphorylation of U1-70K, a constituent of the U1 small nuclear ribonucleoprotein (snRNP), but it is unclear which sites are phosphorylated, and by what enzyme, and how such modification regulates function. By profiling the proteome, we found that the Cdc2-like kinase 1 (CLK1) phosphorylates Ser-226 in the C terminus of U1-70K. This releases U1-70K from subnuclear granules facilitating interaction with U1 snRNP and the serine-arginine (SR) protein SRSF1, critical steps in establishing the 5′ splice site. CLK1 breaks contacts between the C terminus and the RNA recognition motif (RRM) in U1-70K releasing the RRM to bind SRSF1. This reorganization also permits stable interactions between U1-70K and several proteins associated with U1 snRNP. Nuclear induction of the SR protein kinase 1 (SRPK1) facilitates CLK1 dissociation from U1-70K, recycling the kinase for catalysis. These studies demonstrate that CLK1 plays a vital, signal-dependent role in early spliceosomal protein assembly by contouring U1-70K for protein–protein multitasking.

Non-targeting control for MISSION® shRNA library silences SNRPD3 leading to cell death or permanent growth arrest

In parallel with the expansion of RNA interference techniques evidence has been accumulating that RNAi analyses may be seriously biased due to off-target effects of gene-specific shRNAs. Our work points to another possible source of misinterpretations of shRNA-based data – off-target effects of non-targeting shRNA. We found that one such control for the MISSION® library (commercialized TRC library), SHC016, is cytotoxic. Using a lentiviral vector with inducible expression of SHC016 we proved that this shRNA induces apoptosis in murine cells and, depending on p53 status, senescence or mitotic catastrophe in human tumor cell lines. We identified SNRPD3, a core spliceosomal protein, as a major SHC016 target in several cell lines and confirmed in A549 and U251 cell lines that CRISPRi-knockdown of SNRPD3 mimics the effects of SHC016 expression. Our finding disqualifies non-targeting SHC016 shRNA and adds a new premise to the discussion about the sources of uncertainty of RNAi results.

Pan-cancer analysis identifies mutations in SUGP1 that recapitulate mutant SF3B1 splicing dysregulation

The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3′ splice sites (3′ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3′ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3′ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3′ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3′ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.

Spliceosomal Protein Gene BmSPX Regulates Reproductive Organ Development in Bombyx mori

Sex determination and differentiation are nearly universal to all eukaryotic organisms, encompassing diverse systems and mechanisms. Here, we identified a spliceosomal protein gene BmSPX involved in sex determination of the lepidopeteran insect, Bombyx mori. In a transgenic silkworm line that overexpressed the BmSPX gene, transgenic silkworm males exhibited differences in their external genitalia compared to wild-type males, but normal internal genitalia. Additionally, transgenic silkworm females exhibited a developmental disorder of the reproductive organs. Upregulation of BmSPX significantly increased the expression levels of sex-determining genes (BmMasc and BmIMP) and reduced the female-type splice isoform of Bmdsx, which is a key switch gene downstream of the sex-determination pathway. Additionally, co-immunoprecipitation assays confirmed an interaction between the BmSPX protein and BmPSI, an upstream regulatory factor of Bmdsx. Quantitative real-time PCR showed that BmSPX over-expression upregulated the expression of the Hox gene abdominal-B (Adb-B), which is required for specification of the posterior abdomen, external genitalia, and gonads of insects, as well as the genes in the Receptor Tyrosine Kinase (RTK) signaling pathway. In conclusion, our study suggested the involvement of BmSPX, identified as a novel regulatory factor, in the sex-determination pathway and regulation of reproductive organ development in silkworms.