The nonsense-mediated decay (NMD) pathway subjects mRNAs with premature termination codons (PTCs) to rapid decay. The conserved Upf1-3 complex interacts with the eukaryotic translation release factors, eRF3 and eRF1, and triggers NMD when translation termination takes place at a PTC. Contrasting models postulate central roles in PTC-recognition for the exon junction complex in mammals versus the cytoplasmic poly(A)-binding protein (PABP) in other eukaryotes. Here we present evidence for a unified model for NMD, in which PTC recognition in human cells is mediated by a competition between 3' UTR-associated factors that stimulate or antagonize recruitment of the Upf complex to the terminating ribosome. We identify cytoplasmic PABP as a human NMD antagonizing factor, which inhibits the interaction between eRF3 and Upf1 in vitro and prevents NMD in cells when positioned in proximity to the termination codon. Surprisingly, only when an extended 3' UTR places cytoplasmic PABP distally to the termination codon does a downstream exon junction complex enhance NMD, likely through increasing the affinity of Upf proteins for the 3' UTR. Interestingly, while an artificial 3' UTR of >420 nucleotides triggers NMD, a large subset of human mRNAs contain longer 3' UTRs but evade NMD. We speculate that these have evolved to concentrate NMD-inhibiting factors, such as PABP, in spatial proximity of the termination codon.

The drug molecule PTC124 (Ataluren) has been described as a read-through agent, capable of suppressing premature termination codons (PTCs) and restoring functional protein production from genes disrupted by nonsense mutations. Following the discovery of PTC124 there was some controversy regarding its mechanism of action with two reports attributing its activity to an off-target effect on the Firefly luciferase (FLuc) reporter used in the development of the molecule. Despite questions remaining as to its mechanism of action, development of PTC124 continued into the clinic and it is being actively pursued as a potential nonsense mutation therapy. To thoroughly test the ability of PTC124 to read through nonsense mutations, we conducted a detailed assessment comparing the efficacy of PTC124 with the classical aminoglycoside antibiotic read-through agent geneticin (G418) across a diverse range of in vitro reporter assays. We can confirm the off-target FLuc activity of PTC124 but found that, while G418 exhibits varying activity in every read-through assay, there is no evidence of activity for PTC124.

Translation termination at premature termination codons (PTCs) triggers degradation of the aberrant mRNA, but the mechanism by which a termination event is defined as premature is still unclear. Here we show that the physical distance between the termination codon and the poly(A)-binding protein PABPC1 is a crucial determinant for PTC recognition in human cells. "Normal" termination codons can trigger nonsense-mediated mRNA decay (NMD) when this distance is extended; and vice versa, NMD can be suppressed by folding the poly(A) tail into proximity of a PTC or by tethering of PABPC1 nearby a PTC, indicating an evolutionarily conserved function of PABPC1 in promoting correct translation termination and antagonizing activation of NMD. Most importantly, our results demonstrate that spatial rearrangements of the 3' untranslated region can modulate the NMD pathway and thereby provide a novel mechanism for posttranscriptional gene regulation.

Both for understanding mechanisms of disease and for the design of transgenes, it is important to understand the determinants of ribosome velocity, as changes in the rate of translation are important for protein folding, error attenuation, and localization. While there is great variation in ribosomal occupancy along even a single transcript, what determines a ribosome's occupancy is unclear. We examine this issue using data from a ribosomal footprinting assay in yeast. While codon usage is classically considered a major determinant, we find no evidence for this. By contrast, we find that positively charged amino acids greatly retard ribosomes downstream from where they are encoded, consistent with the suggestion that positively charged residues interact with the negatively charged ribosomal exit tunnel. Such slowing is independent of and greater than the average effect owing to mRNA folding. The effect of charged amino acids is additive, with ribosomal occupancy well-predicted by a linear fit to the density of positively charged residues. We thus expect that a translated poly-A tail, encoding for positively charged lysines regardless of the reading frame, would act as a sandtrap for the ribosome, consistent with experimental data.

Pseudogenization is a widespread phenomenon in genome evolution, and it has been proposed to serve as an engine of evolutionary change, especially during human origins (the "less-is-more" hypothesis). However, there has been no comprehensive analysis of human-specific pseudogenes. Furthermore, it is unclear whether pseudogenization itself can be selectively favored and thus play an active role in human evolution. Here we conduct a comparative genomic analysis and a literature survey to identify 80 nonprocessed pseudogenes that were inactivated in the human lineage after its separation from the chimpanzee lineage. Many functions are involved among these genes, with chemoreception and immune response being outstandingly overrepresented, suggesting potential species-specific features in these aspects of human physiology. To explore the possibility of adaptive pseudogenization, we focus on CASPASE12, a cysteinyl aspartate proteinase participating in inflammatory and innate immune response to endotoxins. We provide population genetic evidence that the nearly complete fixation of a null allele at CASPASE12 has been driven by positive selection, probably because the null allele confers protection from severe sepsis. We estimate that the selective advantage of the null allele is about 0.9% and the pseudogenization started shortly before the out-of-Africa migration of modern humans. Interestingly, two other genes related to sepsis were also pseudogenized in humans, possibly by selection. These adaptive gene losses might have occurred because of changes in our environment or genetic background that altered the threat from or response to sepsis. The identification and analysis of human-specific pseudogenes open the door for understanding the roles of gene losses in human origins, and the demonstration that gene loss itself can be adaptive supports and extends the "less-is-more" hypothesis.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.

Tumor heterogeneity is a primary cause of treatment failure and acquired resistance in cancer patients. Even in cancers driven by a single mutated oncogene, variability in response to targeted therapies is well known. The existence of additional genomic alterations among tumor cells can only partially explain this variability. As such, nongenetic factors are increasingly seen as critical contributors to tumor relapse and acquired resistance in cancer. Here, we show that both genetic and nongenetic factors contribute to targeted drug response variability in an experimental model of tumor heterogeneity. We observe significant variability to epidermal growth factor receptor (EGFR) inhibition among and within multiple versions and clonal sublines of PC9, a commonly used EGFR mutant nonsmall cell lung cancer (NSCLC) cell line. We resolve genetic, epigenetic, and stochastic components of this variability using a theoretical framework in which distinct genetic states give rise to multiple epigenetic "basins of attraction," across which cells can transition driven by stochastic noise. Using mutational impact analysis, single-cell differential gene expression, and correlations among Gene Ontology (GO) terms to connect genomics to transcriptomics, we establish a baseline for genetic differences driving drug response variability among PC9 cell line versions. Applying the same approach to clonal sublines, we conclude that drug response variability in all but one of the sublines is due to epigenetic differences; in the other, it is due to genetic alterations. Finally, using a clonal drug response assay together with stochastic simulations, we attribute subclonal drug response variability within sublines to stochastic cell fate decisions and confirm that one subline likely contains genetic resistance mutations that emerged in the absence of drug treatment.