SUMMARY Translation elongation efficiency is largely thought of as the sum of decoding efficiencies for individual codons. Here, we find that adjacent codon pairs modulate translation efficiency. Deploying an approach in Saccharomyces cerevisiae that scored the expression of over 35,000 GFP variants in which three adjacent codons were randomized, we identified 17 pairs of adjacent codons associated with reduced expression. For many pairs, codon order is obligatory for inhibition, implying a more complex interaction than a simple additive effect. Inhibition mediated by adjacent codons occurs during translation itself as GFP expression is restored by increased tRNA levels or by non-native tRNAs with exact-matching anticodons. Inhibition operates in endogenous genes, based on analysis of ribosome profiling data. Our findings suggest translation efficiency is modulated by an interplay between tRNAs at adjacent sites in the ribosome, and that this concerted effect needs to be considered in predicting the functional consequences of codon choice.

The RNA recognition motif (RRM) is the most common RNA-binding domain in eukaryotes. Differences in RRM sequences dictate, in part, both RNA and protein-binding specificities and affinities. We used a deep mutational scanning approach to study the sequence-function relationship of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein (Pab1). By scoring the activity of more than 100,000 unique Pab1 variants, including 1246 with single amino acid substitutions, we delineated the mutational constraints on each residue. Clustering of residues with similar mutational patterns reveals three major classes, composed principally of RNA-binding residues, of hydrophobic core residues, and of the remaining residues. The first class also includes a highly conserved residue not involved in RNA binding, G150, which can be mutated to destabilize Pab1. A comparison of the mutational sensitivity of yeast Pab1 residues to their evolutionary conservation reveals that most residues tolerate more substitutions than are present in the natural sequences, although other residues that tolerate fewer substitutions may point to specialized functions in yeast. An analysis of ∼40,000 double mutants indicates a preference for a short distance between two mutations that display an epistatic interaction. As examples of interactions, the mutations N139T, N139S, and I157L suppress other mutations that interfere with RNA binding and protein stability. Overall, this study demonstrates that living cells can be subjected to a single assay to analyze hundreds of thousands of protein variants in parallel.

Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses, and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here, we have developed ‘enhancer-FACS-Seq’ (eFS) technology for highly parallel identification of active, tissue-specific enhancers in Drosophila embryos. Analysis of enhancers identified by eFS to be active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, novel mesodermal transcription factors (TFs). Naïve Bayes classifiers using TF binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded within them.

Contemporary high-throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs, and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including ChIP sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF cooccupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous cooccupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmdbound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide insights into the transcriptional codes regulating muscle gene expression and offer a generalizable approach for similar studies in other systems.

Cancer stem cells likely survive chemotherapy or radiotherapy by acquiring mutations that inactivate the endogenous apoptotic machinery or by cycling slowly. Thus, knowledge about the mechanisms linking the activation of an alternative cell death modality and the cell cycle machinery could have a transformative impact on the development of new cancer therapies, but the mechanisms remain completely unknown. We investigated the regulation of alternative cell death in Drosophila larval brain neural stem cells (neuroblasts) in which apoptosis is normally repressed. From a screen, we identified two novel loss-of-function alleles of the Cdc20/fizzy (fzy) gene that lead to premature brain neuroblast loss without perturbing cell proliferation in other diploid cell types. Fzy is an evolutionarily conserved regulator of anaphase promoting complex/cyclosome (APC/C). Neuroblasts carrying the novel fzy allele or exhibiting reduced APC/C function display hallmarks of necrosis. By contrast, neuroblasts overexpressing the non-degradable form of canonical APC/C substrates required for cell cycle progression undergo mitotic catastrophe. These data strongly suggest that Fzy can elicit a novel pro-survival function of APC/C by suppressing necrosis. Neuroblasts experiencing catastrophic cellular stress, or overexpressing p53, lose Fzy expression and undergo necrosis. Co-expression of fzy suppresses the death of these neuroblasts. Consequently, attenuation of the Fzydependent survival mechanism functions downstream of catastrophic cellular stress and p53 to eliminate neuroblasts by necrosis. Strategies that target the Fzy-dependent survival mechanism might lead to the discovery of new treatments or complement the pre-existing therapies to eliminate apoptosis-resistant cancer stem cells by necrosis.

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I–HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.