Subtractional Heterogeneity: A Crucial Step toward Defining Specialized Ribosomes.

In this issue of Molecular Cell, Shi et al. (2017) identify translating ribosomes which lack specific proteins and associate with specific classes of mRNAs. This challenges the popular conception of "the ribosome" as a homogeneous, monolithic molecular machine.

A Ribosomopathy Reveals Decoding Defective Ribosomes Driving Human Dysmorphism.

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.

Functional and structural characterization of the chikungunya virus translational recoding signals.

Climate change and human globalization have spurred the rapid spread of mosquito-borne diseases to naïve populations. One such emerging virus of public health concern is chikungunya virus (CHIKV), a member of the Togaviridae family, genus Alphavirus CHIKV pathogenesis is predominately characterized by acute febrile symptoms and severe arthralgia, which can persist in the host long after viral clearance. CHIKV has also been implicated in cases of acute encephalomyelitis, and its vertical transmission has been reported. Currently, no FDA-approved treatments exist for this virus. Recoding elements help expand the coding capacity in many viruses and therefore represent potential therapeutic targets in antiviral treatments. Here, we report the molecular and structural characterization of two CHIKV translational recoding signals: a termination codon read-through (TCR) element located between the nonstructural protein 3 and 4 genes and a programmed -1 ribosomal frameshift (-1 PRF) signal located toward the 3' end of the CHIKV 6K gene. Using Dual-Luciferase and immunoblot assays in HEK293T and U87MG mammalian cell lines, we validated and genetically characterized efficient TCR and -1 PRF. Analyses of RNA chemical modification data with selective 2'-hydroxyl acylation and primer extension (SHAPE) assays revealed that CHIKV -1 PRF is stimulated by a tightly structured, triple-stem hairpin element, consistent with previous observations in alphaviruses, and that the TCR signal is composed of a single large multibulged hairpin element. These findings illuminate the roles of RNA structure in translational recoding and provide critical information relevant for design of live-attenuated vaccines against CHIKV and related viruses.

Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

Activation of the unfolded protein response (UPR) in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus) and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.

Characterization of the metastatic phenotype of a panel of established osteosarcoma cells.

Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. In this study, we collected 18 OS cell lines, confirmed their expression of bone markers and complex karyotypes, and characterized their in vivo tumorgenicity and metastatic potential. Since prior reports included conflicting descriptions of the metastatic and in vivo phenotypes of these models, there was a need for a comparative assessment of metastatic phenotypes using identical procedures in the hands of a single investigative group. We expect that this single characterization will accelerate the study of this metastatic cancer. Using these models we evaluated the expression of six previously reported metastasis-related OS genes. Ezrin was the only gene consistently differentially expressed in all the pairs of high/low metastatic OS cells. We then used a subtractive gene expression approach of the high and low human metastatic cells to identify novel genes that may be involved in OS metastasis. PHLDA1 (pleckstrin homology-like domain, family A) was identified as one of the genes more highly expressed in the high metastatic compared to low metastatic cells. Knocking down PHLDA1 with siRNA or shRNA resulted in down regulation of the activities of MAPKs (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs). Reducing the expression of PHLDA1 also delayed OS metastasis progression in mouse xenograft models.

The ezrin metastatic phenotype is associated with the initiation of protein translation.

We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis.