Visualization of the dynamic interaction between nucleosomal histone H3K9 tri-methylation and HP1α chromodomain in living cells.

Histone lysine methylation is an epigenetic mark that can control gene expression. In particular, H3K9me3 contributes to transcriptional repression by regulating chromatin structure. Successful mitotic progression requires correct timing of chromatin structure changes, including epigenetic marks. However, spatiotemporal information on histone modifications in living cells remains limited. In this study, we created an FRET-based probe for live-cell imaging based on the HP1α chromodomain (HP1αCD), which binds to H3K9me3. The probe was incorporated into chromatin and the emission ratio decreased after treatment with histone methyltransferase inhibitors, indicating that it successfully traced dynamic changes in H3K9me3. Upon entry into mitosis, the probe's emission ratio transiently increased with a concomitant increase in H3K9me3, then exhibited a stepwise decrease, probably due to loss of HP1αCD binding caused by phosphorylation of H3S10 and demethylation of H3K9me3. This probe will be a useful tool for detecting dynamic changes in chromatin structure associated with HP1α.

Splicing modulators elicit global translational repression by condensate-prone proteins translated from introns.

Chemical splicing modulators that bind to the spliceosome have provided an attractive avenue for cancer treatment. Splicing modulators induce accumulation and subsequent translation of a subset of intron-retained mRNAs. However, the biological effect of proteins containing translated intron sequences remains unclear. Here, we identify a number of truncated proteins generated upon treatment with the splicing modulator spliceostatin A (SSA) via genome-wide ribosome profiling and bio-orthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry. A subset of these truncated proteins has intrinsically disordered regions, forms insoluble cellular condensates, and triggers the proteotoxic stress response through c-Jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting the mTORC1 pathway. In turn, this reduces global translation. These findings indicate that creating an overburden of condensate-prone proteins derived from introns represses translation and prevents further production of harmful truncated proteins. This mechanism appears to contribute to the antiproliferative and proapoptotic activity of splicing modulators.

Splicing modulators: on the way from nature to clinic.

Over the course of more than two decades, natural products isolated from various microorganisms and plants have built the foundation for chemical biology research into the mechanism of pre-mRNA splicing. Hand in hand with advances in scientific methodology small molecule splicing modulators have become powerful tools for investigating, not just the splicing mechanism, but also the cellular effect of altered mRNA processing. Based on thorough structure-activity studies, synthetic analogues have moved on from scientific tool compounds to experimental drugs. With current advances in drug discovery methodology and new means of attacking targets previously thought undruggable, we can expect further advances in both research and therapeutics based on small molecule splicing modulators.

Spliceostatin A interaction with SF3B limits U1 snRNP availability and causes premature cleavage and polyadenylation.

RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

Genome-wide Survey of Ribosome Collision.

Ribosome movement is not always smooth and is rather often impeded. For ribosome pauses, fundamental issues remain to be addressed, including where ribosomes pause on mRNAs, what kind of RNA/amino acid sequence causes this pause, and the physiological significance of this attenuation of protein synthesis. Here, we survey the positions of ribosome collisions caused by ribosome pauses in humans and zebrafish using modified ribosome profiling. Collided ribosomes, i.e., disomes, emerge at various sites: Pro-Pro/Gly/Asp motifs; Arg-X-Lys motifs; stop codons; and 3' untranslated regions. The electrostatic interaction between the charged nascent chain and the ribosome exit tunnel determines the eIF5A-mediated disome rescue at the Pro-Pro sites. In particular, XBP1u, a precursor of endoplasmic reticulum (ER)-stress-responsive transcription factor, shows striking queues of collided ribosomes and thus acts as a degradation substrate by ribosome-associated quality control. Our results provide insight into the causes and consequences of ribosome pause by dissecting collided ribosomes.

Targeting the N Terminus of eIF4AI for Inhibition of Its Catalytic Recycling.

DEAD-box ATP-dependent helicases (DEAH/D) are a family of conserved genes predominantly involved in gene expression regulation and RNA processing. As its prototype, eIF4AI is an essential component of the protein translation initiation complex. Utilizing a screening system based on wild-type eIF4AI and its L243G mutant with a changed linker domain, we discovered an eIF4AI inhibitor, sanguinarine (SAN) and used it to study the catalytic mechanism of eIF4AI. Herein, we describe the crystal structure of the eIF4AI-inhibitor complex and demonstrate that the binding site displays certain specificity, which can provide the basis for drug design to target eIF4AI. We report that except for competitive inhibition SAN's possible mechanism of action involves interference with eIF4AI catalytic cycling process by hindering the formation of the closed conformation of eIF4AI. In addition, our results highlight a new targetable site on eIF4AI and confirm eIF4AI as a viable pharmacological target.

Along the Central Dogma-Controlling Gene Expression with Small Molecules.

The central dogma of molecular biology, that DNA is transcribed into RNA and RNA translated into protein, was coined in the early days of modern biology. Back in the 1950s and 1960s, bacterial genetics first opened the way toward understanding life as the genetically encoded interaction of macromolecules. As molecular biology progressed and our knowledge of gene control deepened, it became increasingly clear that expression relied on many more levels of regulation. In the process of dissecting mechanisms of gene expression, specific small-molecule inhibitors played an important role and became valuable tools of investigation. Small molecules offer significant advantages over genetic tools, as they allow inhibiting a process at any desired time point, whereas mutating or altering the gene of an important regulator would likely result in a dead organism. With the advent of modern sequencing technology, it has become possible to monitor global cellular effects of small-molecule treatment and thereby overcome the limitations of classical biochemistry, which usually looks at a biological system in isolation. This review focuses on several molecules, especially natural products, that have played an important role in dissecting gene expression and have opened up new fields of investigation as well as clinical venues for disease treatment.

A gating mechanism for Pi release governs the mRNA unwinding by eIF4AI during translation initiation.

Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation. However, the mechanism of its coupled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release controlled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA unwinding. Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.

Splicing inhibition induces gene expression through canonical NF-κB pathway and extracellular signal-related kinase activation.

Splicing, a process for mRNA maturation, is essential for correct gene expression after transcription. However, recent studies also suggest that splicing affects transcription, but its mechanism remains elusive. We previously reported that treatment with spliceostatin A (SSA), a specific splicing inhibitor targeting the splicing factor SF3b, leads to transcriptional activation of a small subset of genes. To investigate the underlying mechanism we utilized luciferase reporters driven by the Interleukin 8 (IL-8) and cytomegalovirus (CMV) promoters, as both recruit a similar set of transcription factors. We also found that SSA treatment led to increased extracellular signal-regulated protein kinase (ERK) activity and that chemical inhibition of ERK also led to decreased promoter activation. Systematic deletion studies suggested that NF-κB activation is mainly responsible for SSA-induced promoters activation.

Splicing in oncogenesis and tumor suppression.

Post-transcriptional modifications, such as 5' end capping, 3' end polyadenylation and splicing, are necessary for the precise regulation of gene expression and transcriptome integrity. Therefore, it is not surprising that abnormalities of these post-transcriptional modifications prompt numerous diseases, including cancer. In fact, many studies revealed that misregulation of mRNA processing, especially splicing, are observed in a variety of cancer cells. In this review we describe how changes within RNA splicing regulatory elements or mutations in the processing factors alter the expression of tumor suppressors or oncogenes with pathological consequences. In addition, we show how several small molecules that bind to spliceosomal components and splicing regulators inhibit or modulate splicing activity. These compounds have anticancer activity and further development of small molecule modulators has potential in next generation cancer therapy.

Inhibition of eukaryotic translation elongation by the antitumor natural product Mycalamide B.

Mycalamide B (MycB) is a marine sponge-derived natural product with potent antitumor activity. Although it has been shown to inhibit protein synthesis, the molecular mechanism of action by MycB remains incompletely understood. We verified the inhibition of translation elongation by in vitro HCV IRES dual luciferase assays, ribosome assembly, and in vivo [(35)S]methinione labeling experiments. Similar to cycloheximide (CHX), MycB inhibits translation elongation through blockade of eEF2-mediated translocation without affecting the eEF1A-mediated loading of tRNA onto the ribosome, AUG recognition, or dipeptide synthesis. Using chemical footprinting, we identified the MycB binding site proximal to the C3993 28S rRNA residue on the large ribosomal subunit. However, there are also subtle, but significant differences in the detailed mechanisms of action of MycB and CHX. First, MycB arrests the ribosome on the mRNA one codon ahead of CHX. Second, MycB specifically blocked tRNA binding to the E-site of the large ribosomal subunit. Moreover, they display different polysome profiles in vivo. Together, these observations shed new light on the mechanism of inhibition of translation elongation by MycB.

Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin.

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

Garbled messages and corrupted translations.

Following transcription, genomic information begins a long journey toward translation of its nucleotide sequence into the amino acids of a protein. In eukaryotes, synthesized pre-mRNAs become processed to mature mRNAs by 5'-end capping, splicing, 3'-end cleavage and polyadenylation in the nucleus, before being scrutinized for premature stop codons. Each step requires high precision and control to ensure that an intact and readable message is exported to the cytoplasm before finally becoming translated. Two important aspects of these processes are accurately managed by ribonucleoprotein machineries-the spliceosome and the ribosome. Recently, several natural products targeting these macromolecular assemblies have been reported. For the first time in eukaryotes, these molecules allow chemical disruption and dissection of the sophisticated machinery that regulates post-transcriptional events. Beyond their great potential as bioprobes for investigating mRNA regulation and protein synthesis, these compounds also show promise in opening new therapeutic approaches.

Isolation and identification of eukaryotic initiation factor 4A as a molecular target for the marine natural product Pateamine A.

Natural products continue to demonstrate their utility both as therapeutics and as molecular probes for the discovery and mechanistic deconvolution of various cellular processes. However, this utility is dampened by the inherent difficulties involved in isolating and characterizing new bioactive natural products, in obtaining sufficient quantities of purified compound for further biological studies, and in developing bioactive probes. Key to characterizing the biological activity of natural products is the identification of the molecular target(s) within the cell. The marine sponge-derived natural product Pateamine A (PatA) has been found to be an inhibitor of eukaryotic translation initiation. Herein, we describe the methods utilized for identification of the eukaryotic translation initiation factor 4A (eIF4A) as one of the primary protein targets of PatA. We begin by describing the synthesis of an active biotin conjugate of PatA (B-PatA), made possible by total synthesis, followed by its use for affinity purification of PatA binding proteins from cellular lysates. We have attempted to present the methodology as a general technique for the identification of protein targets for small molecules including natural products.

Inhibition of eukaryotic translation initiation by the marine natural product pateamine A.

Translation initiation in eukaryotes is accomplished through the coordinated and orderly action of a large number of proteins, including the eIF4 initiation factors. Herein, we report that pateamine A (PatA), a potent antiproliferative and proapoptotic marine natural product, inhibits cap-dependent eukaryotic translation initiation. PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, yet it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. These results suggest that PatA will be a valuable molecular probe for future studies of eukaryotic translation initiation and may serve as a lead compound for the development of anticancer agents.

Distinct conformational states of nuclear receptor-bound CRSP-Med complexes.

The human CRSP-Med coactivator complex is targeted by a diverse array of sequence-specific regulatory proteins. Using EM and single-particle reconstruction techniques, we recently completed a structural analysis of CRSP-Med bound to VP16 and SREBP-1a. Notably, these activators induced distinct conformational states upon binding the coactivator. Ostensibly, these different conformational states result from VP16 and SREBP-1a targeting distinct subunits in the CRSP-Med complex. To test this, we conducted a structural analysis of CRSP-Med bound to either thyroid hormone receptor (TR) or vitamin D receptor (VDR), both of which interact with the same subunit (Med220) of CRSP-Med. Structural comparison of TR- and VDR-bound complexes (at a resolution of 29 A) indeed reveals a shared conformational feature that is distinct from other known CRSP- Med structures. Importantly, this nuclear receptor-induced structural shift seems largely dependent on the movement of Med220 within the complex.