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). LTM, isomigrastatin and analogs were found to have a potent antiproliferative effect on tumor cell lines and selectively inhibit protein translation. A systematic comparative study of the effects of CHX and LTM on protein translation 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, defining a common binding pocket for both inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.
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.
Highlights d Disome profiling reveals widespread ribosome collisions in vertebrates d Ribosomes are in queues at Pro-Pro/Gly/Asp, Arg-X-Lys, stop codons, and 3 0 UTRs d The positively charged nascent chain weakens the eIF5Amediated rescue of disomes d The stalled disomes on XBP1u mRNA are an endogenous substrate of RQC
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.
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. (Cancer Sci 2012; 103: 1611-1616 mRNA Splicing I n eukaryotes, nascent transcripts (precursor messenger RNA [pre-mRNA]) are subjected to post-transcriptional modifications, including capping of the 5′ end, intron removal by splicing, as well as cleavage and polyadenylation at the 3′ end, to become mature mRNA that are templates for translation.(1) These post-transcriptional modifications are important for efficient gene expression and for the integrity of the transcriptome, hence aberrations in these modifications might perturb gene expression and interfere with vital cellular functions, enabling pathogenesis including carcinogenesis or tumor progression.( 2) Pre-mRNA consist of protein coding regions, exons and intervening sequences, introns.(3) The splicing process joins the exon sequences while removing the introns. These reactions are coordinated and catalyzed by the spliceosome, a large, multi-component ribonuclear complex, consisting of five subcomponent small nuclear ribonucleoprotein particles (snRNPs), named U1, U2, U4, U5 and U6 (Fig. 1). The splicing reactions start with recognition of the intron's 5′ end, the 5′ splice site (5′ ss), by U1 snRNP, followed by SF1 binding to the branch point sequence and interaction of U2AF with the 3′ end of the intron, the 3′ splice site (3′ ss), to form complex E. Complex E turns over to complex A when U2 snRNP replaces SF1. Complex B results from U4/U6•U5 tri-snRNP binding to complex A. In the last step, conformational changes enable two transesterification reactions by which the intron sequence is excised and the adjacent exons joined.This process requires high precision and fidelity as gene expression depends on the integrity of the transcript. A change by only one nucleotide would introduce a frame-shift, which would not only alter the amino acid sequence of the protein but likely introduce a premature termination codon (PTC). Similarly, intron retention by failure to splice will even more likely yield mRNA with a PTC, as intron sequences appear enriched in stop codons. Such mRNA with a PTC are degraded by nonsense mediated d...
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.
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.
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