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Regulation of gene expression involves a wide array of cellular mechanisms that control the abundance of the RNA or protein products of that gene. Here we describe a gene-regulatory mechanism that is based on poly(A) tracks that stall the translation apparatus. We show that creating longer or shorter runs of adenosine nucleotides, without changes in the amino acid sequence, alters the protein output and the stability of mRNA. Sometimes these changes result in the production of an alternative “frame-shifted” protein product. These observations are corroborated using reporter constructs and in the context of recombinant gene sequences. Approximately two percent of genes in the human genome may be subject to this uncharacterized, yet fundamental form of gene regulation. The potential pool of regulated genes encodes many proteins involved in nucleic acid binding. We hypothesize that the genes we identify are part of a large network whose expression is fine-tuned by poly(A)-tracks, and we provide a mechanism through which synonymous mutations may influence gene expression in pathological states.

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Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

A dvanced age, even in healthy individuals, is accompanied by progressive decline of cognitive, metabolic and physiological abilities, and can enhance susceptibility to neurodegenerative, cardiovascular and chronic inflammatory diseases 1,2. Operationally, it is often difficult to determine whether age-associated signatures reflect changes of individual cells or changes in cell-type abundances, especially when performing whole-tissue transcriptional or epigenetic characterization. And even despite a large amount of clinical and epidemiological data 3-7 , we understand very little about the nature of age-associated changes in specific primary cell populations of healthy individuals, particularly with respect to age-associated alterations of the epigenetic landscape. To address this question directly, we focused on classical CD14 + CD16 − monocytes, as they are homogeneous, easily accessible and relatively abundant in blood, which permits multiomics profiling of these cells obtained from a single blood draw. Epigenetic aging can manifest in two key aspects: via age-associated changes in chromatin modifications and in DNA methylation. Robustness of the connection between aging and DNA methylation has been well acknowledged 8-12 ; yet, despite the large number of studies, cell-specific regions of age-associated DNA methylation/demethylation have not been reported so far. Previous studies have predominantly used DNA methylation arrays that detect changes of a predefined set of distant solitary cytosines across the genome 4. This design prevents identification of differentially methylated regions (DMRs), which are expected to be more biologically relevant compared with changes in single isolated CpG sites. In this Article, we used parallel multiomics approaches to characterize intracellular states and extracellular environments of monocytes along healthy aging. To allow for simultaneous identification of continuous age-associated DNA methylation regions and corresponding chromatin context, we utilized enhanced reduced representation bisulfite sequencing (eRRBS) coupled with the ultra-low-input chromatin immunoprecipitation followed by sequencing (ULI-ChIP-seq) 13 approach to profile chromatin modifications from limited input material. Our approach led to the identification of more than 1,000 DMRs, which could not be achieved via methylation array technology. We found no evidence of large-scale remodelling of the chromatin modification landscape along healthy aging, yet revealed distinct chromatin features that were characteristic of age-associated DNA hyper-and hypomethylated regions. Integration of the obtained DMR signatures with

The abundance of messenger RNA (mRNA) is one of the major determinants of protein synthesis. As such, factors that influence mRNA stability often contribute to gene regulation. Polyadenylation of the 3' end of mRNA transcripts, the poly(A) tail, has long been recognized as one of these regulatory elements given its influence on translation efficiency and mRNA stability. Unwanted translation of the poly(A) tail signals to the cell an aberrant polyadenylation event or the lack of stop codons, which makes this sequence an important element in translation fidelity and mRNA surveillance response. Consequently, investigations into the effects of the poly(A) tail lead to the discoveries that poly-lysine as well as other polybasic peptide sequences and, to a much greater extent, polyA mRNA sequences within the open reading frame influence mRNA stability and translational efficiency. Conservation and evolutionary selection of codon usage in polyA track sequences across multiple organisms suggests a biological significance for coding polyA tracks in the regulation of gene expression. Here, we discuss the cellular responses and consequences of coding polyA track translation and synthesis of polybasic peptides. This article is categorized under Translation > Translation Mechanisms Translation > Translation Regulation RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

Tumorigenesis results from the convergence of cell autonomous mutations and corresponding stromal changes that promote tumor cell growth. Senescent cells, which secrete a plethora of pro-tumorigenic factors termed the senescence-associated secretory phenotype (SASP), play an important role in tumor formation. Investigation into SASP regulation revealed that many but not all SASP factors are subject to NF-kB and p38MAPK regulation. However, many pro-tumorigenic SASP factors, including osteopontin (OPN), are not responsive to these canonical pathways leaving the regulation of these factors an open question. We report that the transcription factor c-Myb regulates OPN, IL-6, and IL-8 in addition to 57 other SASP factors. The regulation of OPN is direct as c-Myb binds to the OPN promoter in response to senescence. Further, OPN is also regulated by the known SASP regulator C/EBPβ. In response to senescence, the full-length activating C/EBPβ isoform LAP2 increases binding to the OPN, IL-6, and IL-8 promoters. The importance of both c-Myb and C/EBPβ is underscored by our finding that the depletion of either factor reduces the ability of senescent fibroblasts to promote the growth of preneoplastic epithelial cells.