Clinical actionability enhanced through deep targeted sequencing of solid tumors.

BACKGROUND: Further advances of targeted cancer therapy require comprehensive in-depth profiling of somatic mutations that are present in subpopulations of tumor cells in a clinical tumor sample. However, it is unclear to what extent such intratumor heterogeneity is present and whether it may affect clinical decision-making. To study this question, we established a deep targeted sequencing platform to identify potentially actionable DNA alterations in tumor samples. METHODS: We assayed 515 formalin-fixed paraffin-embedded (FFPE) tumor samples and matched germline DNA (475 patients) from 11 disease sites by capturing and sequencing all the exons in 201 cancer-related genes. Mutations, indels, and copy number data were reported. RESULTS: We obtained a 1000-fold mean sequencing depth and identified 4794 nonsynonymous mutations in the samples analyzed, of which 15.2% were present at <10% allele frequency. Most of these low level mutations occurred at known oncogenic hotspots and are likely functional. Identifying low level mutations improved identification of mutations in actionable genes in 118 (24.84%) patients, among which 47 (9.8%) otherwise would have been unactionable. In addition, acquiring ultrahigh depth also ensured a low false discovery rate (<2.2%) from FFPE samples. CONCLUSIONS: Our results were as accurate as a commercially available CLIA-compliant hotspot panel but allowed the detection of a higher number of mutations in actionable genes. Our study reveals the critical importance of acquiring and utilizing high sequencing depth in profiling clinical tumor samples and presents a very useful platform for implementing routine sequencing in a cancer care institution.

Abba is an essential TRIM/RBCC protein to maintain the integrity of sarcomeric cytoarchitecture.

Organized sarcomeric striations are an evolutionarily conserved hallmark of functional skeletal muscles. Here, we demonstrate that the Drosophila Abba protein, a member of the TRIM/RBCC superfamily, has a pivotal regulatory role in maintaining proper sarcomeric cytoarchitecture during development and muscle usage. abba mutant embryos initially form muscles, but F-actin and Myosin striations become progressively disrupted when the muscles undergo growth and endure increased contractile forces during larval development. Abnormal Myosin aggregates and myofiber atrophy are also notable in the abba mutants. The larval defects result in compromised muscle function, and hence important morphogenetic events do not occur properly during pupation, leading to lethality. Abba is localized at larval Z-discs, and genetic evidence indicates that abba interacts with α-actinin, kettin/D-titin and mlp84B, genes that encode important Z-disc proteins for stable myofibrillar organization and optimal muscle function. RNAi experiments and ultrastructural analysis reveal that Abba has an additional crucial role in sarcomere maintenance in adult muscles. Abba is required to ensure the integrity and function of Z-discs and M-lines. Rescue experiments further show that Abba function is dependent upon its B-box/coiled-coil domain, NHL repeats and RING finger domain. The importance of these presumed protein-protein interactions and ubiquitin ligase-associated domains supports our hypothesis that Abba is needed for specific protein complex formation and stabilization at Z-discs and M-lines.

An RNAi screen identifies additional members of the Drosophila Integrator complex and a requirement for cyclin C/Cdk8 in snRNA 3'-end formation.

Formation of the 3' end of RNA polymerase II-transcribed snRNAs requires a poorly understood group of proteins called the Integrator complex. Here we used a fluorescence-based read-through reporter that expresses GFP in response to snRNA misprocessing and performed a genome-wide RNAi screen in Drosophila S2 cells to identify novel factors required for snRNA 3'-end formation. In addition to the known Integrator complex members, we identified Asunder and CG4785 as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. We also identified a conserved requirement in both fly and human snRNA 3'-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, we observed biochemical association between Integrator proteins and cyclin C/Cdk8, and that overexpression of a kinase-dead Cdk8 causes snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator.

Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB's deadenylation-enhancing and antiproliferative activities.

The mammalian TOB1 and TOB2 proteins have emerged as key players in repressing cell proliferation. Accumulating evidence indicates that TOBs regulate mRNA deadenylation. A recruitment model was proposed in which TOBs promote deadenylation by recruiting CAF1-CCR4 deadenylase complex to the 3' end of mRNAs by simultaneously binding CAF1 and PABP. However, the exact molecular mechanism underlying TOB-promoted deadenylation remains unclear. It is also unclear whether TOBs' antiproliferative and deadenylation-promoting activities are connected. Here, we combine biochemical analyses with a functional assay directly monitoring deadenylation and mRNA decay to characterize the effects of tethering TOBs or their mutant derivatives to mRNAs. The results provide direct evidence supporting the recruitment model and reveal a link between TOBs' antiproliferative and deadenylation-promoting activities. We also find that TOBs' actions in deadenylation are independent of the phosphorylation state of three serines known to regulate antiproliferative actions, suggesting that TOBs arrest cell growth through at least two different mechanisms. TOB1 and TOB2 were interchangeable in the properties tested here, indicating considerable functional redundancy between the two proteins. We propose that their multiple modes of modulating mRNA turnover and arresting cell growth permit the TOB proteins to coordinate their diverse roles in controlling cell growth and differentiation.

A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3'-end formation.

Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3'-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3'-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3'-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3'-end formation and found that processing was strongly dependent upon nucleotides located within the 3' stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3' box. Substitution of the actin promoter for the snRNA promoter abolished proper 3'-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3'-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3'-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.

Messenger RNA half-life measurements in mammalian cells.

The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells.

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5'-to-3' degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

Drosophila mind bomb2 is required for maintaining muscle integrity and survival.

We report that the Drosophila mind bomb2 (mib2) gene is a novel regulator of muscle development. Unlike its paralogue, mib1, zygotic expression of mib2 is restricted to somatic and visceral muscle progenitors, and their respective differentiated musculatures. We demonstrate that in embryos that lack functional Mib2, muscle detachment is observed beginning in mid stage 15 and progresses rapidly, culminating in catastrophic degeneration and loss of most somatic muscles by stage 17. Notably, the degenerating muscles are positive for apoptosis markers, and inhibition of apoptosis in muscles prevents to a significant degree the muscle defects. Rescue experiments with Mib1 and Neuralized show further that these E3 ubiquitin ligases are not capable of ameliorating the muscle mutant phenotype of mib2. Our data suggest strongly that mib2 is involved in a novel Notch- and integrin-independent pathway that maintains the integrity of fully differentiated muscles and prevents their apoptotic degeneration.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

The Drosophila Bruno paralogue Bru-3 specifically binds the EDEN translational repression element.

We reported in our previous work that the EDEN-dependent translational repression of maternal mRNAs was conserved between Drosophila and Xenopus. In Xenopus, this repression is achieved through the binding of EDEN to the Bruno-like factor, EDEN-BP. We show in the present work that the Drosophila Bruno paralogue, the 45 kDa Bru-3 protein (p45), binds specifically to the EDEN element and acts as a homodimer. We describe for the first time a previously undetected 67 amino acid domain, found in the divergent linker region, the lsm domain (lsm stands for linker-specific motif). We propose that the presence of this domain in a subset of the Bruno-like proteins, including Bru-3, EDEN-BP and CUG-BP but not Bruno nor its other paralogue Bru-2, might be responsible for specific RNA recognition. Interestingly, comparative structural analyses using threaders and molecular modelling suggest that the new domain might be distantly related to the first RNA recognition motif of the Drosophila sex-lethal protein (sxl). The phylogenetic analyses and the experimental data based on its specific binding to the EDEN element support the conclusion that Bru-3 is an EDEN-BP/CUG-BP orthologue.

EDEN-dependent translational repression of maternal mRNAs is conserved between Xenopus and Drosophila.

Translational control is a key level in regulating gene expression in oocytes and eggs because many mRNAs are synthesized and stored during oogenesis for latter use at various stages of oocyte maturation and embryonic development. Understanding the molecular mechanisms that underlie this translational control is therefore crucial. Another important issue is the evolutionary conservation of these mechanisms--in other words the determination of their universal and specific aspects. We report here a comparative analysis of a translational repression mechanism that depends on the EDEN (embryo deadenylation element) element. This small cis-acting element, localized in the 3' untranslated region of c-mos and Eg mRNAs, was shown to be involved in a deadenylation process. We demonstrate here that in Xenopus embryos, mRNAs that contain an EDEN are translationally repressed. Next, transgenic flies were used to study the effect of the EDEN motif on translation in Drosophila oocytes. We show that this element also causes the translational repression of a reporter gene in Drosophila demonstrating that the EDEN-dependent translational repression is functionally conserved between Xenopus and Drosophila.