Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation

MicroRNAs (miRNAs) are approximately 22 nucleotide RNAs that negatively regulate the expression of protein-coding genes. In a present model of miRNA function in animals, miRNAs that form imperfect duplexes with their targets inhibit protein expression without affecting mRNA levels. Here, we report that in C. elegans, regulation by the let-7 miRNA results in degradation of its lin-41 target mRNA, despite the fact that its 3'UTR regulatory sequences can only partially base-pair with the miRNA. Furthermore, lin-14 and lin-28 are targets of the lin-4 miRNA, and we show that the mRNA levels for these protein-coding genes significantly decrease in response to lin-4 expression. This study reveals that mRNAs containing partial miRNA complementary sites can be targeted for degradation in vivo, raising the possibility that regulation at the level of mRNA stability may be more common than previously appreciated for the miRNA pathway.     

Uncoupling of lin-14 mRNA and protein repression by nutrient deprivation in Caenorhabditis elegans

In animals, microRNAs (miRNAs), typically, pair to sites of partial complementarity in the 3′-untranslated regions (3′UTRs) of target genes. Regulation by miRNAs often results in down-regulation of target mRNA and protein expression by mechanisms that are yet to be fully elucidated. Additionally, changes in environmental conditions have been shown to influence miRNA function in some cell culture systems. Here, we report the effect of nutrient deprivation on regulation of an endogenous miRNA target in developing worms. In Caenorhabditis elegans, the lin-4 miRNA recognizes multiple sites in the lin-14 3′UTR and directs mRNA degradation and translational repression, but it is unclear how these processes are coupled. In this study, we demonstrate that nutrient deprivation results in loss of lin-14 mRNA, but not protein, repression. In worms removed from feeding conditions, lin-14 mRNA reaccumulates despite the continued expression of lin-4 miRNA. The relative increase in lin-14 mRNA levels during nutrient deprivation is less pronounced in genetic mutants lacking lin-4 miRNA or the lin-14 3′UTR target sites. In conclusion, regulation of lin-14 at the mRNA and protein levels can be uncoupled by changes in culture conditions, indicating that miRNA function can be modulated by environment in multicellular organisms. The awareness that endogenous miRNA pathways can be sensitive to environment is an important consideration for elucidating the mechanism used by miRNAs to regulate target mRNA and protein expression.     

Mathematical modeling of combinatorial regulation suggests that apparent positive regulation of targets by miRNA could be an artifact resulting from competition for mRNA

MicroRNAs bind to and regulate the abundance and activity of target messenger RNA through sequestration, enhanced degradation, and suppression of translation. Although miRNA have a predominantly negative effect on the target protein concentration, several reports have demonstrated a positive effect of miRNA, i.e., increase in target protein concentration on miRNA overexpression and decrease in target concentration on miRNA repression. miRNA–target pair-specific effects such as protection of mRNA degradation owing to miRNA binding can explain some of these effects. However, considering such pairs in isolation might be an oversimplification of the RNA biology, as it is known that one miRNA interacts with several targets, and conversely target mRNA are subject to regulation by several miRNAs. We formulate a mathematical model of this combinatorial regulation of targets by multiple miRNA. Through mathematical analysis and numerical simulations of this model, we show that miRNA that individually have a negative effect on their targets may exhibit an apparently positive net effect when the concentration of one miRNA is experimentally perturbed by repression/overexpression in such a multi-miRNA multitarget situation. We show that this apparent unexpected effect is due to competition and will not be observed when miRNA interact noncompetitively with the target mRNA. This result suggests that some of the observed unusual positive effects of miRNA may be due to the combinatorial complexity of the system rather than due to any inherently unusual positive effect of the miRNA on its target.     

Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state

We have shown previously that transgene expression can be suppressed in hematopoietic cells using vectors that are responsive to microRNA (miRNA) regulation. Here we investigate the potential of this approach for more sophisticated control of transgene expression. Analysis of the relationship between miRNA expression levels and target mRNA suppression suggested that suppression depends on a threshold miRNA concentration. Using this information, we generated vectors that rapidly adjust transgene expression in response to changes in miRNA expression. These vectors sharply segregated transgene expression between closely related states of therapeutically relevant cells, including dendritic cells, hematopoietic and embryonic stem cells, and their progeny, allowing positive/negative selection according to the cells' differentiation state. Moreover, two miRNA target sites were combined to restrict transgene expression to a specific cell type in the liver. Notably, the vectors did not detectably perturb endogenous miRNA expression or regulation of natural targets. The properties of miRNA-regulated vectors should allow for safer and more effective therapeutic applications.     

MicroRNA regulation of molecular networks mapped by global microRNA, mRNA, and protein expression in activated T lymphocytes

MicroRNAs (miRNAs) regulate specific immune mechanisms, but their genome-wide regulation of T lymphocyte activation is largely unknown. We performed a multidimensional functional genomics analysis to integrate genome-wide differential mRNA, miRNA, and protein expression as a function of human T lymphocyte activation and time. We surveyed expression of 420 human miRNAs in parallel with genome-wide mRNA expression. We identified a unique signature of 71 differentially expressed miRNAs, 57 of which were previously not known as regulators of immune activation. The majority of miRNAs are upregulated, mRNA expression of these target genes is downregulated, and this is a function of binding multiple miRNAs (combinatorial targeting). Our data reveal that consideration of this complex signature, rather than single miRNAs, is necessary to construct a full picture of miRNA-mediated regulation. Molecular network mapping of miRNA targets revealed the regulation of activation-induced immune signaling. In contrast, pathways populated by genes that are not miRNA targets are enriched for metabolism and biosynthesis. Finally, we specifically validated miR-155 (known) and miR-221 (novel in T lymphocytes) using locked nucleic acid inhibitors. Inhibition of these two highly upregulated miRNAs in CD4(+) T cells was shown to increase proliferation by removing suppression of four target genes linked to proliferation and survival. Thus, multiple lines of evidence link top functional networks directly to T lymphocyte immunity, underlining the value of mapping global gene, protein, and miRNA expression.     

MicroRNA expression changes following synthesis of three full-sib <Emphasis Type="Italic">Populus</Emphasis> triploid populations with different heterozygosities

Key message

Through high-throughput sequencing, we compared the relative expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one diploid hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. In addition, unbalanced parental expression level dominance of miRNAs were found in the three allotriploid and interspecific hybrid populations, which may reprogram gene expression networks and contribute to the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among one diploid and three triploid hybrid populations, hinting that miRNA abundances do not increase with the genome content. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the slight decrease in miRNA regulation, suggesting an important molecular mechanism of polyploid advantage.

Abstract

Hybridization with three types of induced 2n gametes transmitted different parental heterozygosities has been proven as an efficient method for Populus triploid production. Several researches have shown that miRNA could be non-additively expressed in allopolyploids. However, it is still unclear whether the non-additively expressed miRNAs result from the effect of hybridization or polyploidization, and whether a dose response to the additional genomic content exists for the expression of miRNA. Toward this end, through high-throughput sequencing, we compared the expression levels of miRNA in three full-sib Populus triploid populations with that in their parents and one interspecific hybrid population. We found similar numbers of miRNAs differentially expressed between the parents and the four progeny hybrid populations. Unbalanced parental expression level dominance of miRNAs were found in the three triploid and diploid hybrid populations, which may reprogram gene expression networks and affect the growth of Populus hybrids. These results indicated that hybridization has a great impact on the miRNA expression variation in the newly synthesized Populus triploid and diploid hybrid populations. However, we also found no significant differences in miRNA expression among the three triploid populations and the diploid hybrid population. No dosage effect of miRNA expression could lead to dosage-dependent negative effects on target genes and their downstream pathway in polyploids. We speculate that polyploids may gain advantages from the decrease in miRNA negative regulation, suggesting an important molecular mechanism of polyploid advantage.

     

Molecular architecture of a miRNA-regulated 3' UTR

Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.     

Dimensionless numbers and life history variation in Brown Trout

Summary Dimensionless numbers, made up from components of life history as defined by growth, mortality and maturation, may provide fresh insights into life history evolution. Most studies have previously shown that these numbers are more or less constants within taxa. The variation between taxa may clarify the evolution of different life histories. We examine the variation in three dimensionless numbers using data from 29 populations of Brown TroutSalmo trutta from Norway, and find that the dimensionless numbers are not constants for the Brown Trout populations. We find that the relationship betweenK of the von Bertalanffy growth equation and the mortality rate (M) increased with increasing growth rate. Also, relative length at maturity (L /L _inf) increased with increasing asymptotic length (L _inf). We suggest that more such data should be collected from a large number of species and taxonomic groups, to allow a more detailed assessment of why these dimensionless numbers appear to be constants in some taxa and not in others.