An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.

Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states-quiescence and cell cycling-can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. In support of this mechanism, analysis of cells at a point before cell cycle entry (i.e., before the G1/S transition) revealed a p21-CDK2 axis that determines quiescent and cycling cell states. Our findings suggest a mechanistic role for p21 in generating heterogeneity in both normal tissues and tumors.tumor heterogeneity | cell dormancy | synthetic uORF | nongenetic cell heterogeneity | positive feedback loop

The initiation of mRNA translation from start codons other than AUG was previously believed to be rare and of relatively low impact. More recently, evidence has suggested that as much as half of all translation initiation utilizes non-AUG start codons, codons that deviate from AUG by a single base. Furthermore, non-AUG start codons have been shown to be involved in regulation of expression and disease etiology. Yet the ability to gauge expression based on the sequence of a translation initiation site (start codon and its flanking bases) has been limited. Here we have performed a comprehensive analysis of translation initiation sites that utilize non-AUG start codons. By combining genetic-reporter, cell-sorting, and high-throughput sequencing technologies, we have analyzed the expression associated with all possible variants of the -4 to +4 positions of non-AUG translation initiation site motifs. This complete motif analysis revealed that 1) with the right sequence context, certain non-AUG start codons can generate expression comparable to that of AUG start codons, 2) sequence context affects each non-AUG start codon differently, and 3) initiation at non-AUG start codons is highly sensitive to changes in the flanking sequences. Complete motif analysis has the potential to be a key tool for experimental and diagnostic genomics.

Acute muscle injury and physiological stress from chronic muscle diseases and aging lead to impairment of skeletal muscle function. This raises the question of whether p53, a cellular stress sensor, regulates muscle tissue repair under stress conditions. By investigating muscle differentiation in the presence of genotoxic stress, we discovered that p53 binds directly to the myogenin promoter and represses transcription of myogenin, a member of the MyoD family of transcription factors that plays a critical role in driving terminal muscle differentiation. This reduction of myogenin protein is observed in G1-arrested cells and leads to decreased expression of late but not early differentiation markers. In response to acute genotoxic stress, p53-mediated repression of myogenin reduces post-mitotic nuclear abnormalities in terminally differentiated cells. This study reveals a mechanistic link previously unknown between p53 and muscle differentiation, and suggests new avenues for managing p53-mediated stress responses in chronic muscle diseases or during muscle aging. Cell Death and Differentiation (2015) 22, 560-573; doi:10.1038/cdd.2014; published online 12 December 2014The tumor suppressor p53 promotes cell cycle arrest or apoptosis in response to diverse stress signals such as DNA damage, thus preventing propagation of genetically compromised cells. [1][2][3] Among the diverse functions attributed to p53, a growing body of evidence supports its role in regulation of differentiation and maintenance of cellular function and integrity. 1,[4][5][6][7] For example, p53 represses Nanog to maintain genetic stability of the stem cell pool by promoting differentiation of mouse embryonic stem cells (mESCs) after DNA damage. 6 Skeletal muscle differentiation, a key step during muscle tissue formation, is orchestrated by the MyoD family of myogenic regulatory factors (MRFs). MyoD determines the myogenic lineage, whereas myogenin, a member of the MRF family, functions downstream of MyoD and plays a critical role in driving terminal differentiation as myogenin-null mice show a lethal deficiency of differentiated skeletal muscle. [8][9][10][11][12][13] The dynamic differentiation program of skeletal muscle is characterized by the orderly expression of genes and structural changes that can be recapitulated in vitro, as myogenic cells undergo cell cycle withdrawal and express early and then late differentiation genes with the formation of mononucleated myocytes to elongated multinucleated myotubes. 8,9,14 Under normal unstressed conditions, p53 has been shown to promote muscle differentiation in vitro. [15][16][17][18][19][20] In contrast, under stress-associated conditions such as inflammation, chronic exposure to double-strand DNA breaks, and aging, enhanced p53 activity has been shown to correlate with skeletal muscle atrophy in vivo. [21][22][23][24][25] In addition, it has been shown that p53 and its downstream effectors are required for an inflammatory cytokine-mediated inhibition of myogenic differentiation in vitro. 22 Int...

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