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Background Next-generation sequencing (NGS) informs many biological questions with unprecedented depth and nucleotide resolution. These assays have created a need for analytical tools that enable users to manipulate data nucleotide-by-nucleotide robustly and easily. Furthermore, because many NGS assays encode information jointly within multiple properties of read alignments ― for example, in ribosome profiling, the locations of ribosomes are jointly encoded in alignment coordinates and length ― analytical tools are often required to extract the biological meaning from the alignments before analysis. Many assay-specific pipelines exist for this purpose, but there remains a need for user-friendly, generalized, nucleotide-resolution tools that are not limited to specific experimental regimes or analytical workflows. Results Plastid is a Python library designed specifically for nucleotide-resolution analysis of genomics and NGS data. As such, Plastid is designed to extract assay-specific information from read alignments while retaining generality and extensibility to novel NGS assays. Plastid represents NGS and other biological data as arrays of values associated with genomic or transcriptomic positions, and contains configurable tools to convert data from a variety of sources to such arrays. Plastid also includes numerous tools to manipulate even discontinuous genomic features, such as spliced transcripts, with nucleotide precision. Plastid automatically handles conversion between genomic and feature-centric coordinates, accounting for splicing and strand, freeing users of burdensome accounting. Finally, Plastid’s data models use consistent and familiar biological idioms, enabling even beginners to develop sophisticated analytical workflows with minimal effort. Conclusions Plastid is a versatile toolkit that has been used to analyze data from multiple NGS assays, including RNA-seq, ribosome profiling, and DMS-seq. It forms the genomic engine of our ORF annotation tool, ORF-RATER, and is readily adapted to novel NGS assays. Examples, tutorials, and extensive documentation can be found at https://plastid.readthedocs.io .

We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5′ untranslated regions in cancer, and expose new targets for therapeutic intervention. The translation of upstream open reading frames in skin tumour models protects some cancer-related mRNAs from global reductions in protein synthesis during the early stages of tumour initiation, suggesting that unconventional translation has a crucial role in tumorigenesis. Deregulated translation and tumour progression Elaine Fuchs and colleagues uncover a role for the translation of upstream open reading frames (uORFs)—gene-expression regulatory elements present in many messenger RNAs—in skin tumour models. This oncogene-driven shift in translation shields some pro-tumorigenic mRNAs from global reductions in protein synthesis during the early stages of tumorigenesis, suggesting that tumour drivers may use uORF translation to enact oncogenic transformation.

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. For a gene to give rise to a protein, its DNA is first used as a template to produce a messenger RNA molecule. Each group of three nucleotides within the messenger RNA encodes an amino acid, and structures called ribosomes assemble the protein by joining together amino acids in the correct order. The nucleotide triplets are called codons, and some are known as stop codons because they typically instruct the ribosome to stop adding amino acids. Sometimes ribosomes interpret stop codons as amino acid insertion signals, giving rise to an extended protein with a modified structure or function. This phenomenon is known as stop codon readthrough, and is required for many viruses to complete their reproductive cycles. However, much less is known about stop codon readthrough in other organisms. Now, Dunn et al. have used a technique called ribosome profiling to analyze stop codon readthrough across the entire genome of the fruit fly Drosophila melanogaster. An enzyme was used to fragment messenger RNA, and those fragments that were specifically engaged by ribosomes—and thus likely to encode protein—were sequenced. Stop codon readthrough occurred much more often than had been expected based on previous studies. Indeed, computational analysis strongly suggests that evolution has favored this process for certain fruit fly genes. Moreover, stop codon readthrough was also observed in yeast and human cells, suggesting that it is important in many organisms, not just the fruit fly. Stop codon readthrough thus provides a novel way for organisms to tune the expression levels and functions of their genes, both throughout the lifetime of an individual, and the evolution of a species.

Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.

When the school board refused to relent-even though the district provided similar state-mandated opt-outs for sex-ed instruction in health classes, including in high school-the parents sued, requesting an injunction forbidding the school district from implementing the policy while the case was being litigated. Justice Stephen Breyer predicted in his dissent that the majority's reasoning would eventually compel the court to require states to approve religious charter schools (an issue the court is also taking up this term in St. Isidore of Seville Catholic Virtual School v. Drummond) and even vouchers, asking if \"the State must pay parents for the religious equivalent of the secular benefit provided.\" In the lower courts, the school board had argued that if parents object to the curriculum they can send their children to private school.