MicroRNAs can generate thresholds in target gene expression

MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that repress gene expression in a sequence-dependent manner. We performed single-cell measurements using quantitative fluorescence microscopy and flow cytometry to monitor a target gene's protein expression in the presence and absence of regulation by miRNA. We find that although the average level of repression is modest, in agreement with previous population-based measurements, the repression among individual cells varies dramatically. In particular, we show that regulation by miRNAs establishes a threshold level of target mRNA below which protein production is highly repressed. Near this threshold, protein expression responds sensitively to target mRNA input, consistent with a mathematical model of molecular titration. These results show that miRNAs can act both as a switch and as a fine-tuner of gene expression.     

The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA

Retention of juvenile traits in the adult reproductive phase characterizes a process known as neoteny, and speculation exists over whether it has contributed to the evolution of new species. The dominant Corngrass1 (Cg1) mutant of maize is a neotenic mutation that results in phenotypes that may be present in the grass-like ancestors of maize. We cloned Cg1 and found that it encodes two tandem miR156 genes that are overexpressed in the meristem and lateral organs. Furthermore, a target of Cg1 is teosinte glume architecture1 (tga1), a gene known to have had a role in the domestication of maize from teosinte. Cg1 mutant plants overexpressing miR156 have lower levels of mir172, a microRNA that targets genes controlling juvenile development. By altering the relative levels of both microRNAs, it is possible to either prolong or shorten juvenile development in maize, thus providing a mechanism for how species-level heterochronic changes can occur in nature.     

Target mimicry provides a new mechanism for regulation of microRNA activity

MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.     

Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster

Human adenocarcinomas commonly harbor mutations in the KRAS and MYC proto-oncogenes and the TP53 tumor suppressor gene. All three genetic lesions are potentially pro-angiogenic, as they sustain production of vascular endothelial growth factor (VEGF). Yet Kras-transformed mouse colonocytes lacking p53 formed indolent, poorly vascularized tumors, whereas additional transduction with a Myc-encoding retrovirus promoted vigorous vascularization and growth. In addition, VEGF levels were unaffected by Myc, but enhanced neovascularization correlated with downregulation of anti-angiogenic thrombospondin-1 (Tsp1) and related proteins, such as connective tissue growth factor (CTGF). Both Tsp1 and CTGF are predicted targets for repression by the miR-17-92 microRNA cluster, which was upregulated in colonocytes coexpressing K-Ras and c-Myc. Indeed, miR-17-92 knockdown with antisense 2'-O-methyl oligoribonucleotides partly restored Tsp1 and CTGF expression; in addition, transduction of Ras-only cells with a miR-17-92-encoding retrovirus reduced Tsp1 and CTGF levels. Notably, miR-17-92-transduced cells formed larger, better-perfused tumors. These findings establish a role for microRNAs in non-cell-autonomous Myc-induced tumor phenotypes.     

Parallel bacterial evolution within multiple patients identifies candidate pathogenicity genes

Bacterial pathogens evolve during the infection of their human host(1-8), but separating adaptive and neutral mutations remains challenging(9-11). Here we identify bacterial genes under adaptive evolution by tracking recurrent patterns of mutations in the same pathogenic strain during the infection of multiple individuals. We conducted a retrospective study of a Burkholderia dolosa outbreak among subjects with cystic fibrosis, sequencing the genomes of 112 isolates collected from 14 individuals over 16 years. We find that 17 bacterial genes acquired nonsynonymous mutations in multiple individuals, which indicates parallel adaptive evolution. Mutations in these genes affect important pathogenic phenotypes, including antibiotic resistance and bacterial membrane composition and implicate oxygen-dependent regulation as paramount in lung infections. Several genes have not previously been implicated in pathogenesis and may represent new therapeutic targets. The identification of parallel molecular evolution as a pathogen spreads among multiple individuals points to the key selection forces it experiences within human hosts.     

Mechanisms and convergence of compensatory evolution in mammalian mitochondrial tRNAs

The function of protein and RNA molecules depends on complex epistatic interactions between sites. Therefore, the deleterious effect of a mutation can be suppressed by a compensatory second-site substitution. In relating a list of 86 pathogenic mutations in human tRNAs encoded by mitochondrial genes to the sequences of their mammalian orthologs, we noted that 52 pathogenic mutations were present in normal tRNAs of one or several nonhuman mammals. We found at least five mechanisms of compensation for 32 pathogenic mutations that destroyed a Watson-Crick pair in one of the four tRNA stems: restoration of the affected Watson-Crick interaction (25 cases), strengthening of another pair (4 cases), creation of a new pair (8 cases), changes of multiple interactions in the affected stem (11 cases) and changes involving the interaction between the loop and stem structures (3 cases). A pathogenic mutation and its compensating substitution are fixed in a lineage in rapid succession, and often a compensatory interaction evolves convergently in different clades. At least 10%, and perhaps as many as 50%, of all nucleotide substitutions in evolving mammalian tRNAs participate in such interactions, indicating that the evolution of tRNAs proceeds along highly epistatic fitness ridges.     

Functional classification of drugs by properties of their pairwise interactions

Multidrug treatments are increasingly important in medicine and for probing biological systems. Although many studies have focused on interactions between specific drugs, little is known about the system properties of a full drug interaction network. Like their genetic counterparts, two drugs may have no interaction, or they may interact synergistically or antagonistically to increase or suppress their individual effects. Here we use a sensitive bioluminescence technique to provide quantitative measurements of pairwise interactions among 21 antibiotics that affect growth rate in Escherichia coli. We find that the drug interaction network possesses a special property: it can be separated into classes of drugs such that any two classes interact either purely synergistically or purely antagonistically. These classes correspond directly to the cellular functions affected by the drugs. This network approach provides a new conceptual framework for understanding the functional mechanisms of drugs and their cellular targets and can be applied in systems intractable to mutant screening, biochemistry or microscopy.