Interchangeable SF3B1 inhibitors interfere with pre-mRNA splicing at multiple stages

The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1''s role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.     

A dynamic bulge in the U6 RNA internal stem-loop functions in spliceosome assembly and activation

The highly conserved internal stem-loop (ISL) of U6 spliceosomal RNA is unwound for U4/U6 complex formation during spliceosome assembly and reformed upon U4 release during spliceosome activation. The U6 ISL is structurally similar to Domain 5 of group II self-splicing introns, and contains a dynamic bulge that coordinates a Mg++ ion essential for the first catalytic step of splicing. We have analyzed the causes of growth defects resulting from mutations in the Saccharomyces cerevisiae U6 ISL-bulged nucleotide U80 and the adjacent C67-A79 base pair. Intragenic suppressors and enhancers of the cold-sensitive A79G mutation, which replaces the C-A pair with a C-G pair, suggest that it stabilizes the ISL, inhibits U4/U6 assembly, and may also disrupt spliceosome activation. The lethality of mutations C67A and C67G results from disruption of base-pairing potential between U4 and U6, as these mutations are fully suppressed by compensatory mutations in U4 RNA. Strikingly, suppressor analysis shows that the lethality of the U80G mutation is due not only to formation of a stable base pair with C67, as previously proposed, but also another defect. A U6-U80G strain in which mispairing with position 67 is prevented grows poorly and assembles aberrant spliceosomes that retain U1 snRNP and fail to fully unwind the U4/U6 complex at elevated temperatures. Our data suggest that the U6 ISL bulge is important for coupling U1 snRNP release with U4/U6 unwinding during spliceosome activation.     

Network theory reveals principles of spliceosome structure and dynamics

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DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.     

Histone modifying enzymes and cancer: going beyond histones

Mutations in the molecular pathways that regulate cell proliferation, differentiation, and cell death all contribute to cancer formation. Enzymes that covalently modify histones affect these pathways by controlling the dynamic remodeling of chromatin structure. This article reviews several connections between histone modifying enzymes and cancer that are likely mediated via both histone and non-histone substrates. We propose that multiple protein modifications, including phosphorylation, methylation, and acetylation, cross regulate one another to coordinate intermolecular signaling, and that miscues in this regulation can lead to oncogenesis.     

Rice histone deacetylase genes display specific expression patterns and developmental functions

Histone deacetylases (HDAC) are important in plant gene expression. Here we show that the expression of rice HDAC genes is both tissue/organ-specific, and most of them are responsive to drought or salt stresses. Over-expression of several rice HDACs did not produce any visible phenotype, whereas down-regulation of a few HDAC genes affected different developmental aspects. Specifically, down-regulation of HDA703 by amiRNA reduced rice peduncle elongation and fertility, while inactivation of a closely related homolog HDA710 by RNAi affected vegetative growth. HDA704 RNAi altered plant height and flag leaf morphology. Down-regulation of HDT702 led to the production of narrowed leaves and stems. These data suggest that rice HDAC genes may have divergent developmental functions compared with closely related homologs in Arabidopsis.     

Design,synthesis and evaluation of antiproliferative activity of melanoma-targeted histone deacetylase inhibitors

The clinical validation of histone deacetylase inhibition as a cancer therapeutic modality has stimulated interest in the development of new generation of potent and tumor selective histone deacetylase inhibitors (HDACi). With the goal of selective delivery of the HDACi to melanoma cells, we incorporated the benzamide, a high affinity melanin-binding template, into the design of HDACi to generate a new series of compounds 10a-b and 11a-b which display high potency towards HDAC1 and HDAC6. However, these compounds have attenuated antiproliferative activities relative to the untargeted HDACi. An alternative strategy furnished compound 14, a prodrug bearing the benzamide template linked via a labile bond to a hydroxamate-based HDACi. This pro-drug compound showed promising antiproliferative activity and warrant further study.     

Yersinia pestis acetyltransferase-mediated dual acetylation at the serine and lysine residues enhances the auto-ubiquitination of ubiquitin ligase MARCH8 in human cells

Lysine acetylation is known as a post translational modification (PTM) by histone acetyltransferases (HAT) that modifies histones and non-histone proteins to regulate gene expression. Serine acetylation, however, is reported in mammalian hosts by serine acetyltransferase of Yersinia pestis (YopJ) during infection. The protein target and cellular function of bacterial YopJ in mammalian systems are not fully addressed. Here we report dual acetylation at the serine and lysine residues by transiently expressed serine acetyltransferase YopJ mimicking Y. pestis infection in HeLa cells. Using shotgun proteomics followed by label-free quantification, we demonstrate an increase of dual acetylation in YopJ transfected human cells, including 10 Ser- (YopJ/non-YopJ 1.3-fold, p = 0.02) and 8 Lys- (YopJ/non-YopJ 3.5-fold, p = 0.00003) acetylation sites. Specifically, YopJ expression augments acetylation of membrane-associated E3 ubiquitin ligase MARCH8 at the serine residue Sac44, Sac71 and Sac253, and the lysine residue Kac247 and Kac252. YopJ-mediated Ser- and Lys-acetylation of MARCH8 is further confirmed by Western blotting using the specific antibodies against MARCH8 Sac71 and pan-acetyl lysine. Functional study demonstrates that YopJ-mediated Ser- and Lys-acetylation affects the auto-ubiquitination of MARCH8. The mutant C172A of YopJ previously shown to abolish the acetyltransferase activity also reduces Ser- and Lys-acetylation and diminishes the auto-ubiquitination of MARCH8. In support, MARCH8 is indeed acetylated at serine and lysine in vitro by purified YopJ but the activity is reduced by the C172A mutant in YopJ. Our study provides evidence that bacterial serine acetyltransferase YopJ mediates Ser- and Lys-acetylation and affects auto-ubiquitination of ubiquitin ligase MARCH8 in human cells.     

Structure and function of an RNase H domain at the heart of the spliceosome

Precursor-messenger RNA (pre-mRNA) splicing encompasses two sequential transesterification reactions in distinct active sites of the spliceosome that are transiently established by the interplay of small nuclear (sn) RNAs and spliceosomal proteins. Protein Prp8 is an active site component but the molecular mechanisms, by which it might facilitate splicing catalysis, are unknown. We have determined crystal structures of corresponding portions of yeast and human Prp8 that interact with functional regions of the pre-mRNA, revealing a phylogenetically conserved RNase H fold, augmented by Prp8-specific elements. Comparisons to RNase H-substrate complexes suggested how an RNA encompassing a 5'-splice site (SS) could bind relative to Prp8 residues, which on mutation, suppress splice defects in pre-mRNAs and snRNAs. A truncated RNase H-like active centre lies next to a known contact region of the 5'SS and directed mutagenesis confirmed that this centre is a functional hotspot. These data suggest that Prp8 employs an RNase H domain to help assemble and stabilize the spliceosomal catalytic core, coordinate the activities of other splicing factors and possibly participate in chemical catalysis of splicing.     

Inhibition of histone acetylation and deacetylation enzymes affects longevity,development, and fecundity in the pea aphid (Acyrthosiphon pisum)

Histone acetylation is an evolutionarily conserved epigenetic mechanism of eukaryotic gene regulation which is tightly controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In insects, life-history traits such as longevity and fecundity are severely affected by the suppression of HAT/HDAC activity, which can be achieved by RNA-mediated gene silencing or the application of chemical inhibitors. We used both experimental approaches to investigate the effect of HAT/HDAC inhibition in the pea aphid (Acyrthosiphon pisum) a model insect often used to study complex life-history traits. The silencing of HAT genes (kat6b, kat7, and kat14) promoted survival or increased the number of offspring, whereas targeting rpd3 (HDAC) reduced the number of viviparous offspring but increased the number of premature nymphs, suggesting a role in embryogenesis and eclosion. Specific chemical inhibitors of HATs/HDACs showed a remarkably severe impact on life-history traits, reducing survival, delaying development, and limiting the number of offspring. The selective inhibition of HATs and HDACs also had opposing effects on aphid body weight. The suppression of HAT/HDAC activity in aphids by RNA interference or chemical inhibition revealed similarities and differences compared to the reported role of these enzymes in other insects. Our data suggest that gene expression in A. pisum is regulated by multiple HATs/HDACs, as indicated by the fitness costs triggered by inhibitors that suppress several of these enzymes simultaneously. Targeting multiple HATs or HDACs with combined effects on gene regulation could, therefore, be a promising approach to discover novel targets for the management of aphid pests.     

Brain metastasis is predetermined in early stages of cutaneous melanoma by CD44v6 expression through epigenetic regulation of the spliceosome

Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell‐surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM development. The expression of CD44v6 also enhances the migration of MBM cells by hyaluronic acid and hepatocyte growth factor exposure. Additionally, CD44v6‐positive MBM migration is reduced by blocking with a CD44v6‐specific monoclonal antibody or knocking down CD44v6 by siRNA. ESRP1 and ESRP2 splicing factors correlate with CD44v6 expression in PRM, and ESRP1 knockdown significantly decreases CD44v6 expression. However, an epigenetic silencing of ESRP1 is observed in metastatic melanoma, specifically in MBM. In advanced melanomas, CD44v6 expression correlates with PTBP1 and U2AF2 splicing factors, and PTBP1 knockdown significantly decreases CD44v6 expression. Overall, these findings open a new avenue for understanding the high affinity of melanoma to progress to MBM, suggesting CD44v6 as a potential MBM‐specific factor with theranostic utility for stratifying patients.     

Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B(act) spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosome's catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.