Mimicking of splicing-related retinitis pigmentosa mutations in C. elegans allow drug screens and identification of disease modifiers.

CRISPR/Cas and the high conservation of the spliceosome components facilitate the mimicking of human pathological mutations in splicing factors of model organisms. The degenerative retinal disease retinitis pigmentosa (RP) is caused by mutations in distinct types of genes, including missense mutations in splicing factors that provoke RP in an autosomal dominant form (s-adRP). Using CRISPR in Caenorhabditis elegans, we generated mutant strains to mimic s-adRP mutations reported in PRPF8 and SNRNP200. Whereas these inherited mutations are present in heterozygosis in patients, C. elegans allows the maintenance of these mutations as homozygotes, which is advantageous for genetic and drug screens. We found that snrp-200(cer23[V676L]) and prp-8(cer14[H2302del]) display pleiotropic phenotypes, including reduced fertility. However, snrp-200(cer24[S1080L]) and prp-8(cer22[R2303G]) are weak alleles suitable for RNAi screens for identifying genetic interactions, which could uncover potential disease modifiers. We screened a collection of RNAi clones for splicing-related genes and identified three splicing factors: isy-1/ISY1, cyn-15/PPWD1 and mog-2/SNRPA1, whose partial inactivation may modify the course of the disease. Interestingly, these three genes act as modifiers of prp-8(cer22) but not of snrp-200(cer24). Finally, a screen of the strong allele prp-8(cer14) with FDA-approved drugs did not identify molecules capable of alleviating the temperature-sensitive sterility. Instead, we detected drugs, such as dequalinium chloride, which exacerbated the phenotype, and therefore, are potentially harmful to s-adRP patients since they may accelerate the progression of the disease.

Discovery of spiro[indole-3,2'-pyrrolidin]-2(1H)-one based inhibitors targeting Brr2, a core component of the U5 snRNP.

Bad response to refrigeration 2 (Brr2) is a member of the Ski2-like RNA helicases, and an essential component of the U5 small nuclear ribonucleoprotein (snRNP). A particularly important role of Brr2 is the ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step in spliceosomal activation. Despite its biological importance, selective inhibitor for Brr2 had not been reported until our recent report. Here, we describe novel and structurally distinct spiro[indole-3,2'-pyrrolidin]-2(1H)-one based Brr2 inhibitors with superior activity to the previously reported 4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione series. Using an RNA dependent ATPase assay as a guide, high-throughput screening, hit validation by structure-activity relationship (SAR) study, and subsequent chemical optimization to increase the ATPase inhibitory activity were performed. Thereafter, selectivity and helicase inhibitory activity of optimized compounds were confirmed. In the course of the study, compounds were synthesized using a three-component reaction, which accelerated the optimization process. All these efforts finally culminated in the discovery of the potent and selective Brr2 inhibitors (32a and 33a) exhibiting helicase inhibitory activity at submicromolar concentrations. Thus, compounds 32a and 33a could be valuable molecular probes to study the functions of Brr2 and molecular machinery of RNA splicing.

Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos.

Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope (NE), where they facilitate exchange of molecules between the cytoplasm and the nucleoplasm. In most cell types, NPCs are evenly distributed around the NE. However, the mechanisms dictating NPC distribution are largely unknown. Here, we used the model organism Caenorhabditis elegans to identify genes that affect NPC distribution during early embryonic divisions. We found that down-regulation of the Sm proteins, which are core components of the spliceosome, but not down-regulation of other splicing factors, led to clustering of NPCs. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. We further found that these mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.

Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylation and deacetylation.

The removal of intervening sequences from a primary RNA transcript is catalyzed by the spliceosome, a large complex consisting of five small nuclear (sn) RNAs and more than 150 proteins. At the start of the splicing cycle, the spliceosome assembles anew onto each pre-mRNA intron in an ordered process. Here, we show that several small-molecule inhibitors of protein acetylation/deacetylation block the splicing cycle: by testing a small number of bioactive compounds, we found that three small-molecule inhibitors of histone acetyltransferases (HATs), as well as three small-molecule inhibitors of histone deacetylases (HDACs), block pre-mRNA splicing in vitro. By purifying and characterizing the stalled spliceosomes, we found that the splicing cycle is blocked at distinct stages by different inhibitors: two inhibitors allow only the formation of A-like spliceosomes (as determined by the size of the stalled complexes and their snRNA composition), while the other compounds inhibit activation for catalysis after incorporation of all U snRNPs into the spliceosome. Mass-spectrometric analysis of affinity-purified stalled spliceosomes indicated that the intermediates differ in protein composition both from each other and from previously characterized native A and B splicing complexes. This suggests that the stalled complexes represent hitherto unobserved intermediates of spliceosome assembly.

Identification of small molecules that mitigate vincristine-induced neurotoxicity while sensitizing leukemia cells to vincristine.

Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose-limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR-induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human-induced pluripotent stem cell-derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR's antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.

Analysis of Genetically Regulated Gene Expression Identifies a Prefrontal PTSD Gene, SNRNP35, Specific to Military Cohorts.

To reveal post-traumatic stress disorder (PTSD) genetic risk influences on tissue-specific gene expression, we use brain and non-brain transcriptomic imputation. We impute genetically regulated gene expression (GReX) in 29,539 PTSD cases and 166,145 controls from 70 ancestry-specific cohorts and identify 18 significant GReX-PTSD associations corresponding to specific tissue-gene pairs. The results suggest substantial genetic heterogeneity based on ancestry, cohort type (military versus civilian), and sex. Two study-wide significant PTSD associations are identified in European and military European cohorts; ZNF140 is predicted to be upregulated in whole blood, and SNRNP35 is predicted to be downregulated in dorsolateral prefrontal cortex, respectively. In peripheral leukocytes from 175 marines, the observed PTSD differential gene expression correlates with the predicted differences for these individuals, and deployment stress produces glucocorticoid-regulated expression changes that include downregulation of both ZNF140 and SNRNP35. SNRNP35 knockdown in cells validates its functional role in U12-intron splicing. Finally, exogenous glucocorticoids in mice downregulate prefrontal Snrnp35 expression.

Positive regulation of ASK1-mediated c-Jun NH(2)-terminal kinase signaling pathway by the WD-repeat protein Gemin5.

Gemin5 is a 170-kDa WD-repeat-containing protein that was initially identified as a component of the survival of motor neurons (SMN) complex. We now show that Gemin5 facilitates the activation of apoptosis signal-regulating kinase 1 (ASK1) and downstream signaling. Gemin5 physically interacted with ASK1 as well as with the downstream kinases SEK1 and c-Jun NH(2)-terminal kinase (JNK1), and it potentiated the H(2)O(2)-induced activation of each of these kinases in intact cells. Moreover, Gemin5 promoted the binding of ASK1 to SEK1 and to JNK1, as well as the ASK1-induced activation of JNK1. In comparison, Gemin5 did not physically associate with MKK7, MKK3, MKK6, or p38. Furthermore, depletion of endogenous Gemin5 by RNA interference (RNAi) revealed that Gemin5 contributes to the activation of ASK1 and JNK1, and to apoptosis induced by H(2)O(2) and tumor necrosis factor-alpha (TNFalpha) in HeLa cells. Together, our results suggest that Gemin5 functions as a scaffold protein for the ASK1-JNK1 signaling module and thereby potentiates ASK1-mediated signaling events.

Splicing regulator SLU7 is essential for maintaining liver homeostasis.

A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.

Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8.

The Ski2-like RNA helicase Brr2 is a core component of the spliceosome that must be tightly regulated to ensure correct timing of spliceosome activation. Little is known about mechanisms of regulation of Ski2-like helicases by protein cofactors. Here we show by crystal structure and biochemical analyses that the Prp8 protein, a major regulator of the spliceosome, can insert its C-terminal tail into Brr2's RNA-binding tunnel, thereby intermittently blocking Brr2's RNA-binding, adenosine triphosphatase, and U4/U6 unwinding activities. Inefficient Brr2 repression is the only recognizable phenotype associated with certain retinitis pigmentosa-linked Prp8 mutations that map to its C-terminal tail. Our data show how a Ski2-like RNA helicase can be reversibly inhibited by a protein cofactor that directly competes with RNA substrate binding.

The U11-48K protein contacts the 5' splice site of U12-type introns and the U11-59K protein.

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5' splice sites (5'ss). This interaction did not require branch point recognition and was sensitive to 5'ss mutations, suggesting that 48K interacts with the 5'ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5'ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5'ss, as well as functionally important interactions within the U11/U12 di-snRNP.

PP2B and PP1alpha cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling.

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV- or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca2+)-calmodulin-protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1alpha (PP1alpha) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1alpha releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca2+ signaling, PP2B and PP1alpha alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription.

snRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN.

Splicing snRNPs (small nuclear ribonucleoproteins) are essential sub-units of the spliceosome. Here we report the establishment of stable cell lines expressing fluorescently tagged SmB, a core snRNP protein. Analysis of these stable cell lines has allowed us to characterize the nuclear pathway that leads to snRNP accumulation in nuclear speckles and has identified a limiting nucleolar step in the pathway that can be saturated by overexpression of Sm proteins. After nuclear import, newly assembled snRNPs accumulate first in a subset of Cajal bodies that contain both p80-coilin and the survival of motor neurons protein (SMN) and not in bodies that contain p80-coilin but lack SMN. Treatment of cells with leptomycin B (LMB) inhibits both the accumulation of snRNPs in nuclear bodies and their subsequent accumulation in speckles. The formation of Cajal bodies is enhanced by Sm protein expression and the assembly of new snRNPs. Formation of heterokaryons between HeLa cell lines expressing Sm proteins and primary cells that usually lack Cajal bodies results in the detection of Cajal bodies in primary cell nuclei. Transient over-expression of exogenous SmB alone is sufficient to induce correspondingly transient Cajal body formation in primary cells. These data indicate that the level of snRNP protein expression and snRNP assembly, rather than the expression levels of p80-coilin or SMN, may be a key trigger for Cajal body formation.