Uncovering New Functions for MicroRNAs in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

In the race to understand microRNA (miRNA) functions in development and physiology, Caenorhabditis elegans investigators were the first out of the gate with the cloning and analysis of the lin-4 and let-7 miRNAs [1,2]. The starting point of strong, penetrant loss of function phenotypes facilitated these advancements. However, subsequent functional analysis of miRNAs in C. elegans was hampered by the lack of easily observable loss-of-function phenotypes [3]. There are several possible models to account for this observation. First, redundancy between related miRNAs can account for the absence of phenotypes in mutants missing individual miRNA genes [4,5]. Second, miRNAs may also function redundantly with unrelated miRNAs or other regulatory mechanisms. Third, identification of miRNA functions may require the analysis of specific cells during development, assays typically not included in initial broad phenotypic analyses. For example, the lsy-6 miRNA is an essential regulator of a chemosensory neuron cell fate in C. elegans [6]. Such a specialized function would not have been identified in broad phenotypic analyses. Finally, miRNAs may act to ‘fine-tune’ gene expression, to maintain protein levels of targets in an optimal range. Loss of this relatively minor regulatory input by miRNAs would not be expected to result in penetrant, observable defects under normal conditions. Recent work has analyzed the functions of individual miRNAs under conditions of environmental or physiological stress. With these approaches, functions for individual miRNAs, which remain elusive under normal growth conditions, have been uncovered. These stresses can be introduced through genetic mutations, environmental perturbations, or through the normal aging process. These results are consistent with the hypothesis that miRNAs act to ensure the robustness of developmental or physiological pathways [7]

The \u3cem\u3emir-51\u3c/em\u3e Family of MicroRNAs Functions in Diverse Regulatory Pathways in \u3cem\u3eCaenorhbditis elegans\u3c/em\u3e

The mir-51 family of microRNAs (miRNAs) in C. elegans are part of the deeply conserved miR-99/100 family. While loss of all six family members (mir-51-56) in C. elegans results in embryonic lethality, loss of individual mir-51 family members results in a suppression of retarded developmental timing defects associated with the loss of alg-1. The mechanism of this suppression of developmental timing defects is unknown. To address this, we characterized the function of the mir-51 family in the developmental timing pathway. We performed genetic analysis and determined that mir-51 family members regulate the developmental timing pathway in the L2 stage upstream of hbl-1. Loss of the mir-51 family member, mir-52, suppressed retarded developmental timing defects associated with the loss of let-7 family members and lin-46. Enhancement of precocious defects was observed for mutations in lin-14, hbl-1, and mir-48(ve33), but not later acting developmental timing genes. Interestingly, mir-51 family members showed genetic interactions with additional miRNA-regulated pathways, which are regulated by the let-7 and mir-35 family miRNAs, lsy-6, miR-240/786, and miR-1. Loss of mir-52 likely does not suppress miRNA-regulated pathways through an increase in miRNA biogenesis or miRNA activity. We found no increase in the levels of four mature miRNAs, let-7, miR-58, miR-62 or miR-244, in mir-52 or mir-52/53/54/55/56 mutant worms. In addition, we observed no increase in the activity of ectopic lsy-6 in the repression of a downstream target in uterine cells in worms that lack mir-52. We propose that the mir-51 family functions broadly through the regulation of multiple targets, which have not yet been identified, in diverse regulatory pathways in C. elegans

Functional Analysis of MicroRNA Pathway Genes in the Somatic Gonad and Germ Cells During Ovulation in \u3cem\u3eC. Elegans\u3c/em\u3e

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in animal development and physiology, though functions for most miRNAs remain unknown. Worms with reduced miRNA biogenesis due to loss of Drosha or Pasha/DGCR8 activity are sterile and fail to ovulate, indicating that miRNAs are required for the process of oocyte maturation and ovulation. Starting with this penetrant sterile phenotype and using new strains created to perform tissue specific RNAi, we characterized the roles of the C. elegans Pasha, pash-1, and two miRNA-specific Argonautes, alg-1 and alg-2, in somatic gonad cells and in germ cells in the regulation of ovulation. Conditional loss of pash-1activity resulted in a reduced rate of ovulation and in basal and ovulatory sheath contractions. Similarly, knockdown of miRNA-specific Argonautes in the cells of the somatic gonad by tissue-specific RNAi results in a reduction of the ovulation rate and in basal and ovulatory sheath contractions. Reduced miRNA pathway gene activity resulted in a range of defects, including oocytes that were pinched upon entry of the oocyte into the distal end of the spermatheca in about 42% of the ovulation events observed following alg-1 RNAi. This phenotype was not observed on worms exposed to control RNAi. In contrast, knockdown of alg-1 and alg-2 in germ cells results in few defects in oocyte maturation and ovulation. These data identify specific steps in the process of ovulation that require miRNA pathway gene activity in the somatic gonad cells

Fluctuations and noise in cancer development

This paper explores fluctuations and noise in various facets of cancer development. The three areas of particular focus are the stochastic progression of cells to cancer, fluctuations of the tumor size during treatment, and noise in cancer cell signalling. We explore the stochastic dynamics of tumor growth and response to treatment using a Markov model, and fluctutions in tumor size in response to treatment using partial differential equations. We also explore noise within gene networks in cancer cells, and noise in inter-cell signalling.Comment: 11 pages, 6 figure

Stage-Specific Timing of the microRNA Regulation of \u3cem\u3elin-28\u3c/em\u3e by the Heterochronic Gene \u3cem\u3elin-14\u3c/em\u3e in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

In normal development, the order and synchrony of diverse developmental events must be explicitly controlled. In the nematode Caenorhabditis elegans, the timing of larval events is regulated by hierarchy of proteins and microRNAs (miRNAs) known as the heterochronic pathway. These regulators are organized in feedforward and feedback interactions to form a robust mechanism for specifying the timing and execution of cell fates at successive stages. One member of this pathway is the RNA binding protein LIN-28, which promotes pluripotency and cell fate decisions in successive stages. Two genetic circuits control LIN-28 abundance: it is negatively regulated by the miRNA lin-4, and positively regulated by the transcription factor LIN-14 through a mechanism that was previously unknown. In this report, we used animals that lack lin-4 to elucidate LIN-14’s activity in this circuit. We demonstrate that three let-7 family miRNAs—miR-48, miR-84, and miR-241—inhibit lin-28 expression. Furthermore, we show genetically that these miRNAs act between lin-14 and lin-28, and that they comprise the pathway by which lin-14 positively regulates lin-28. We also show that the lin-4 family member mir-237, also regulates early cell fates. Finally, we show that the expression of these miRNAs is directly inhibited by lin-14 activity, making them the first known targets of lin-14 that act in the heterochronic pathway

Loss of Individual MicroRNAs Causes Mutant Phenotypes in Sensitized Genetic Backgrounds in \u3cem\u3eC. elegans\u3c/em\u3e

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate the translation and/or stability of their mRNA targets. Previous work showed that for most miRNA genes of C. elegans, single-gene knockouts did not result in detectable mutant phenotypes. This may be due, in part, to functional redundancy between miRNAs. However, in most cases, worms carrying deletions of all members of a miRNA family do not display strong mutant phenotypes. They may function together with unrelated miRNAs or with non-miRNA genes in regulatory networks, possibly to ensure the robustness of developmental mechanisms. To test this, we examined worms lacking individual miRNAs in genetically sensitized backgrounds. These include genetic backgrounds with reduced processing and activity of all miRNAs or with reduced activity of a wide array of regulatory pathways. With these two approaches, we identified mutant phenotypes for 25 out of 31 miRNAs included in this analysis. Our findings describe biological roles for individual miRNAs and suggest that the use of sensitized genetic backgrounds provides an efficient approach for miRNA functional analysis

miR-786 Regulation of a Fatty-Acid Elongase Contributes to Rhythmic Calcium-Wave Initiation in \u3cem\u3eC. elegans\u3c/em\u3e

Background: Rhythmic behaviors are ubiquitous phenomena in animals. In C. elegans, defecation is an ultradian rhythmic behavior: every ∼50 s a calcium wave initiating in the posterior intestinal cells triggers the defecation motor program that comprises three sequential muscle contractions. Oscillatory calcium signaling is central to the periodicity of defecation. The posteriormost intestinal cells function as the pacemaker for this rhythmic behavior, although it is unclear how the supremacy of these cells for calcium-wave initiation is controlled. Results: We describe how the loss of the mir-240/786 microRNA cluster, which results in arrhythmic defecation, causes ectopic intestinal calcium-wave initiation. mir-240/786 expression in the intestine is restricted to the posterior cells that function as the defecation pacemaker. Genetic data indicate that mir-240/786 functions upstream of the inositol 1,4,5-trisphosphate (IP3) receptor. Through rescue analysis, it was determined that miR-786 functions to regulate defecation. Furthermore, we identified elo-2, a fatty-acid elongase with a known role in defecation cycling, as a direct target for miR-786. We propose that the regulation of palmitate levels through repression of elo-2 activity is the likely mechanistic link to defecation. Conclusions: Together, these data indicate that miR-786 confers pacemaker status on posterior intestinal cells for the control of calcium-wave initiation through the regulation of elo-2 and, subsequently, palmitate levels. We propose that a difference in fatty-acid composition in the posterior intestinal cells may alter the activities of membrane proteins, such as IP3-receptor or TRPM channels, that control pacemaker activity in the C. elegans intestine

The \u3cem\u3elet-7\u3c/em\u3e MicroRNA Family Members \u3cem\u3emir\u3c/em\u3e-48, \u3cem\u3emir\u3c/em\u3e-84, and mir-241 Function Together to Regulate Developmental Timing in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3′ UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions

A directional wave measurement attack against the Kish key distribution system

The Kish key distribution system has been proposed as a classical alternative to quantum key distribution. The idealized Kish scheme elegantly promises secure key distribution by exploiting thermal noise in a transmission line. However, we demonstrate that it is vulnerable to nonidealities in its components, such as the finite resistance of the transmission line connecting its endpoints. We introduce a novel attack against this nonideality using directional wave measurements, and experimentally demonstrate its efficacy.Lachlan J. Gunn, Andrew Allison & Derek Abbot

Most \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e MicroRNAs are Individually Not Essential for Development or Viability

MicroRNAs (miRNAs), a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs