Victor R Ambros et al. (2003) International Worm Meeting "MicroRNAs and other tiny endogenous RNAs in C. elegans"

Ann Lavanway, Christopher M Hammell, Rosalind C Lee, Victor R Ambros, Peter T Williams, David Jewell

[International Worm Meeting, 2003]

MicroRNAs (miRNAs) are very small (~22 nt long) noncoding RNAs that are generated from hairpin precursor transcripts by Dicer-dependent processing. miRNAs likely inhibit translation of mRNAs via imprecise antisense base-pairing. Small interfering RNAs (siRNAs) are similar in size to miRNAs, but they recognize targets by precise complementarity, and elicit RNA-mediated interference (RNAi). To identify as many of the C. elegans miRNA genes as possible, and other kinds of ~22 nt endogenous RNAs expressed in C. elegans, we employed phylogenetic comparisons of noncoding genomic sequences, and sequencing of size-selected cDNA libraries. Among the new small RNAs detected are 21 distinct miRNA sequences that were not previously described in C. elegans. We estimate the size of the C. elegans miRNA gene family to be somewhat more than 100 distinct gene; about 80% of these are conserved in a C. briggsae, and about 30% have apparent homologs in insects and/or vertebrates. We also identified 33 distinct members of a second class of small RNA, which we call tiny noncoding RNAs (tncRNAs). tncRNAs are similar in length to miRNAs, but unlike miRNAs, they are apparently not processed from short hairpin precursors. However, like miRNAs, tncRNAs are transcribed from noncoding genomic sequence, and in some cases, exhibit developmentally regulated expression. tncRNAs also resemble miRNAs in their lack of precise complementarity to any worm mRNA. Therefore, tncRNAs could act in a fashion exemplified by the canonical miRNAs, lin-4 and let-7, and recognize RNA targets through imprecise antisense base-pairing. Finally, we identified a third class of ~22 nt RNAs in C. elegans that are produced directly from protein coding sequences and are predicted to have precise antisense complementarity to messenger RNAs. These apparent endogenous siRNAs represent sequences from more than 500 different protein-coding genes, including prominently transposons, kinases, transcription factors, and F-box proteins. Preliminary analysis of worms deficient in Dicer, alg-1/alg-2 or rde- genes indicates that the accumulation of miRNAs, tncRNAs and siRNAs depend on distinct pathways. These results suggest that RNA-mediated gene regulation and gene silencing may be broadly deployed in normal worm cells through the action of diverse classes of small RNAs.

McJunkin, Katherine et al. (2015) International Worm Meeting "A regulatory module involving a microRNA and an RNA binding protein controls sex determination and dosage compensation in the C. elegans embryo."

Ambros, Victor, McJunkin, Katherine

[International Worm Meeting, 2015]

MicroRNAs control gene expression through binding to complementary target mRNAs. Although the roles of microRNAs in C. elegans larval development have been extensively studied, relatively little is understood about the function of microRNAs in embryonic development. The mir-35-41 microRNA cluster is expressed maternally and in embryos, and is essential for embryonic development and fecundity. Here, we show that the mir-35-41 microRNA cluster promotes hermaphrodite sex determination and dosage compensation via regulation of its target gene sup-26.We observed that many transcripts are abberantly upregulated in mir-35-41(nDf50) mutant embryos, but that these transcripts are not enriched for mir-35-41 target sites. We hypothesized that mir-35-41 might indirectly repress these transcripts by promoting the function of a transcriptional repressor. By analyzing publicly-available gene expression data, we found that the transcriptional changes observed in mir-35-41(nDf50) are highly similar to those observed in dosage compensation mutant embryos (sdc-2(lf)). Additionally, mir-35-41(nDf50) strongly enhances her-1(gf) masculinization, another hallmark of compromised dosage compensation. We sought a target gene that might link mir-35-41 to dosage compensation. SUP-26 is an RNA-binding protein which was cloned as a suppressor of her-1(gf) masculinization, and is a predicted mir-35-41 target gene. Sup-26(lf) is epistatic to mir-35-41(nDf50);her-1(gf) masculinization and to the derepression of dosage-compensated genes observed in mir-35-41(nDf50) embryos. Thus, the dosage compensation phenotypes of mir-35-41(nDf50) are dependent on SUP-26, supporting a role for SUP-26 downstream of mir-35-41 in regulating dosage compensation.Finally, we performed high-throughput sequencing-crosslinking immunoprecipitation (HITS-CLIP) to define the targets of SUP-26 binding. Through this method, we identified >800 RNA peaks of direct SUP-26 binding. Nearly all of the SUP-26-bound regions are in the 3'UTRs of protein coding genes (>94% of peaks). Among the targets of SUP-26 binding are members of the dosage compensation complex, providing a putative link between mir-35-41, SUP-26, and dosage compensation. Overall, this work provides surprising new insight into the role that microRNAs play in regulating embryonic gene expression, and draws the first link between microRNAs and dosage compensation. .

McJunkin, Katherine et al. (2013) International Worm Meeting "The mir-35 family of microRNAs regulates hermaphrodite fecundity, male development, and genetically interacts with the sex determination pathway."

Ambros, Victor, McJunkin, Katherine

[International Worm Meeting, 2013]

The mir-35-42 family is one of three known essential microRNA families in C. elegans. Deletion of all eight microRNAs in the family results in embryonic lethality. To better understand the function of the mir-35 family, we have examined a viable strain in which seven of eight family members are deleted (mir-35-41), resulting in temperature-dependent embryonic lethality. First, we further characterized the mir-35-41 phenotype, and observed that mir-35-41 also exhibits a temperature-dependent defect in hermaphrodite fecundity, and males display abnormal tail morphology and reduced mating efficiency. We hypothesized that proteins that bind specifically to the mir-35-RNA-induced silencing complex (Wu, et al.) may be modulators of mir-35 family activity in the context of these phenotypes. Thus we examined genetic interactions between these candidate genes and the sensitized mir-35-41 background. In particular, inactivation of sup-26 strongly enhanced mir-35-41 lethality, suggesting that SUP-26 may act as a positive co-factor for the remaining mir-35 family member (mir-42). Paradoxically, sup-26 mRNA bears a conserved mir-35 family target site. If sup-26 is aberrantly up-regulated in mir-35 family mutants, then sup-26 inactivation should suppress the mir-35-41 phenotype. Indeed, a weak sup-26 allele partially suppresses the temperature-sensitive defect in fecundity of mir-35-41 (while enhancing embryonic lethality). Thus, sup-26 may have a dual role as a positive mir-35 family regulator and a mir-35 target gene. SUP-26 is an RNA-binding protein that plays a masculinizing role in both hermaphrodites and males through translational inhibition of tra-2 mRNA. tra-2 does not contain a conserved mir-35 binding site, and tra-2 loss of function does not suppress mir-35-41 embryonic lethality. However, in addition to sup-26(lf), other feminizing mutations, such as tra-2(gf) or fem-3(lf) also enhance lethality of mir-35-41. Our future work aims to elucidate whether SUP-26 indeed regulates the activity of mir-35-42 or other microRNA families, and to gain a clearer understanding of how the mir-35 family functionally interacts with the sex determination pathway.

Nelson, Charles et al. (2017) International Worm Meeting "Post-transcriptional regulation of the microRNA let-7 in C. elegans."

Ambros, Victor, Nelson, Charles

[International Worm Meeting, 2017]

let-7 is a microRNA whose sequence, function, and regulation are well conserved throughout the animal kingdom. LIN-28 is an RNA binding protein that negative regulates let-7 biogenesis. In mammals, biochemical and crystal structure studies show that LIN-28 achieves this negative regulation by binding the let-7/let-7* stem loop of either primary-let-7 or pre-let-7. Interestingly, the post-transcriptional repression that LIN-28 imposes upon let-7 biogenesis is conserved in C. elegans, but the stem-loop sequence is not. Recent studies have demonstrated that in C. elegans LIN-28 binds to the primary-let-7 transcript downstream of the let-7/let-7* stem loop to repress let-7 biogenesis. In addition to LIN-28 repression, two other mechanisms of post-transcriptional regulation of let-7 biogenesis have also been described. First, let-7 complimentary sequences in its primary transcript have been described to promote its own biogenesis. Second, trans-splicing of pri-let-7 has been shown to remodel its secondary structure to facilitate processing. This work aims to further characterize how these various modes of post-transcriptional control of let-7 biogenesis are integrated to achieve robustly regulated expression of let-7 in the context of the heterochronic pathway.

McJunkin, Katherine et al. (2011) International Worm Meeting "Genetic identification of post-transcriptional modulators of microRNAs."

Ambros, Victor, McJunkin, Katherine

[International Worm Meeting, 2011]

MicroRNAs are large class of small noncoding RNAs that regulate the expression of protein-coding genes in plants and animals. By repressing target mRNAs, microRNAs play important roles throughout normal development and in diverse processes in differentiated cell types. Accordingly, individual microRNAs display highly specific expression patterns, often restricted to a particular developmental stage or tissue. MicroRNA expression patterns are partially derived from transcriptional regulation; however, many examples of post-transcriptional regulation of microRNAs have also recently been described. All steps of the microRNA biogenesis pathway appear to be regulated in certain contexts in animals, including Drosha cleavage of the primary transcript and Dicer cleavage of the pre-microRNA. In conjunction with biogenesis, the regulated turnover of mature microRNAs (or pri- or pre-microRNA intermediates) could contribute to their complex spatio-temporal expression patterns. In comparison to biogenesis, very little is understood about the effectors or regulators of microRNA degradation. Finally, the activity of microRNA silencing complexes can be enhanced or dampened by the binding of accessory factors that do not affect microRNA processing or stability. The aim of this research is to elucidate novel modes of post-transcriptional control of microRNAs in animals, focusing on mechanisms of microRNA degradation. The accumulation of mature let-7 and mir-35 and mir-51 family members are developmentally controlled at a post-transcriptional level. Thus, transgenic worms have been generated that bear fluorescent reporters to directly monitor changes in the activity of these microRNA families by simultaneously tracking microRNA transcription and target repression. Genetic screens are ongoing in these backgrounds to identify mutants in which the normal developmental patterns of target repression are disrupted, to identify genes that impact microRNA processing or turnover.

Ow, Maria et al. (2011) International Worm Meeting "The FLH family of transcription factors is epistatic with the insulin/IGF-1 signaling pathway and modulates L1 diapause and stress resistance in C. elegans."

Ow, Maria, Ambros, Victor

[International Worm Meeting, 2011]

Under unfavorable environmental conditions, many organisms are able to suspend development until more favorable conditions are restored. C. elegans embryos hatched under conditions lacking any food enter an arrested stage where development ceases during the first larval stage (L1 diapause) and resumes growth only when nutrition becomes available. The insulin/IGF-1 signaling pathway is a key regulator of L1 diapause, aging and stress resistance. Mutations in members of this signaling pathway, such as the insulin-like tyrosine kinase receptor, daf-2, or its main effector, the FOXO transcription factor daf-16, exhibit aberrant lifespan and responses to environmental and physiological stresses. A severe temperature sensitive daf-2 mutant, daf-2(e979), arrests at the first larval stage at the elevated temperature even in the presence of food, suggesting that the developmental pathways for feeding or the sensing of nutritional cues are defective in this mutant. We have previously shown that a family of Zn-finger transcription factors, FLH-1, FLH-2, and FLH-3, repress the expression of several microRNAs including lin-4, mir-48 and mir-241 during embryogenesis. Mutations in members of this family result in pleiotropic phenotypes including L1 larval lethality, egg-laying defects, premature expression of post-embryonic microRNAs during embryogenesis, and L1 diapause in the presence of food. Here we report that the FLH transcription factors, along with the insulin/IGF-1 signaling pathway, modulate L1 diapause and stress resistance and are therefore involved in feeding and/or nutrient sensing and in the maintenance of the animal during physiological and environmental stresses.

Ren, Zhiji et al. (2013) International Worm Meeting "Involvement of C. elegans let-7-Family developmental timing microRNAs in bacterial pathogen response."

Ren, Zhiji, Ambros, Victor

[International Worm Meeting, 2013]

C. elegans let-7 family (let-7-Fam) microRNAs (let-7, mir-48, mir-241 and mir-84) function semi-redundantly to regulate the developmental timing of stage-specific events in hypodermal seam cell lineages. We observed that upon infection of C. elegans larvae with human opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa), the developmental timing defects of certain let-7-Fam mutants are enhanced. This enhancement is mediated by the p38 MAPK innate immunity pathway, though interestingly the upstream TIR domain adaptor protein TIR-1 seems to be bypassed. The enhancement of let-7-Fam developmental timing defects by P. aeruginosa infection implies that the activity of one or more let-7-Fam microRNAs may be decreased in response to P. aeruginosa, perhaps to enhance the animal's antibacterial response. Consistent with this supposition, we observed that let-7(mg279) animals exhibit an improved survival against P. aeruginosa infection. Interestingly, let-7(mg279) animals are more resistant to P. aeruginosa even though they accumulate pathogen in their intestine more rapidly than wild type. mir-48(0); mir-84(0) and mir-48(0), mir-241(0) double mutants display a complex, two-phase survival curve on P. aeruginosa, suggesting possible positive and negative roles of these let-7-Fam microRNAs in pathogen resistance. Upon P. aeruginosa infection, we observed slight decreases in the levels of certain let-7-Fam microRNAs and an increase in LIN-28 protein level. Consistent with a possible role for lin-28 in modulating pathogen response via let-7, we observed that lin-28(lf) animals exhibit an hypersensitivity to P. aeruginosa, and this hypersensitivity can be suppressed by let-7(0). This suggests that post-transcriptional regulation by LIN-28, as well as transcriptional regulation by p38 MAPK responsive factors, may contribute to a down-regulation of let-7-Fam microRNA activity in response to P. aeruginosa infection. By exploring these dual functions of let-7-Fam microRNAs in pathogen response as well as developmental timing, we aim to achieve a better understanding of how these microRNAs are integrated into broader gene regulatory networks that confer robustness to developmental events against environmental stresses.

Thatcher, Elizabeth J et al. (2011) International Worm Meeting "Sensitized Backgrounds Reveal Critical Roles for microRNA Families."

Ambros, Victor, Thatcher, Elizabeth J

[International Worm Meeting, 2011]

MicroRNAs (miRNAs) are a class of small regulatory RNAs that have been implicated in the control of many cellular functions including cell specification, differentiation, proliferation, and metabolism. A smaller number of miRNAs are expressed in stem cells and at early stages of development, including some that are thought to maintain pluripotency. Interestingly, even though many C. elegans miRNAs are highly conserved, for many single deletion knockouts or even family knockouts, phenotypes are subtle or not evident. C. elegans have 23 miRNA families, 9 of which are conserved through humans. The Horvitz group showed that most miRNAs are not essential for development or viability on their own. This indicates a high degree of functional redundancy among miRNA family members, and between unrelated miRNA families in the worm. Recent findings from the Abbott group illustrated that sensitizing the worm may reveal several mutant phenotypes associated with loss of individual miRNAs or families. I examined the phenotypes associated with several critical miRNA effector complexes to determine whether combining miRNA family knockouts were able to alleviate or exacerbate the observed phenotypes. For instance, RNAi of Alg-1 or Alg-2 produces characteristic developmental delays and disruptions that include problems with molting, slow growth, absent or missing alae, and protruding vulva. These abnormalities are occasionally severe enough to produce embryonic or larval lethality. Examining the effects of Alg-1 or Alg-2 RNAi on miRNA family mutants produces an interesting picture. Most notably, the loss of some miRNAs appears to alleviate the impact of RNAi on Alg-1 or Alg-2 to produce a less severe phenotype as compared to RNAi on wild-type N2 animals. Moreover, some family mutants are uniquely sensitive to either Alg-1 or Alg-2 RNAi. These experiments should illuminate how microRNA pathways are integrated with broader gene regulatory networks, and are adapted to control the robust expression of diverse developmental events.

Thatcher, Elizabeth J et al. (2013) International Worm Meeting "Acute, High-throughput RNAi in C. elegans."

Ambros, Victor, Thatcher, Elizabeth J

[International Worm Meeting, 2013]

C. elegans have previously demonstrated a robust ability to utilize exogenous dsRNA to silence endogenous genes and transgenes by RNA interference (RNAi). Typically, dsRNA is delivered for RNAi by microinjection, soaking in naked dsRNA, or feeding of bacteria expressing dsRNA. Microinjection requires training and expensive equipment and is usually applied to relatively small numbers of worms. Soaking and feeding are more amenable to higher throughput, although soaking requires a larger quantity of dsRNA, and both feeding and soaking can involve a relatively long time course between application of the dsRNA and when knockdown is observed. We have developed a method for the use of electroporation to efficiently deliver dsRNA to C. elegans larvae or adults for silencing of somatic or germline genes. Electroporation offers a number of significant advantages over previous methods for RNAi in C. elegans. Electroporation results in rapid delivery of dsRNA to populations of worms without expensive equipment or training. Electroporation requires a lower concentration of dsRNA than does soaking, and results in more rapid knockdown than does feeding or soaking. Electroporation is most effective at long-term knockdown when using long dsRNA rather than short (25 base pairs) dicer substrate RNA or siRNA (22 base pairs, with 2 nt 3' overhangs). Interestingly, short dsRNAs and siRNAs result in acute but transient knockdown (for example of a GFP transgene), whereas long dsRNA results in long-term (and heritable) knockdown. Long dsRNA can be easily in vitro transcribed from widely available RNAi plasmids. Electroporation can also be completed using dsRNA extracted from bacteria expressing RNAi plasmids. This allows for efficient screening of RNAi libraries using a 96-well format. Thus, electroporation can be easily employed for rapid and either transient or long-term knockdown of endogenous genes or transgenes on a high-throughput scale.

Harandi, Omid F. et al. (2011) International Worm Meeting "Heterochronic genes and Wnt pathway components regulate asymmetric division and cell fate of epidermal stem cells in C. elegans."

Harandi, Omid F., Ambros, Victor

[International Worm Meeting, 2011]

Aberrant regulation of spatio-temporal signals may cause the improper cell fate specification of stem/progenitor cells. Asymmetric cell division, and the underlying cell polarity, is a defining property of stem cell self-renewal. Loss of polarity leads to symmetric division, resulting in hyperproliferation of stem cells. Stem cells have been shown to switch between symmetric and asymmetric cell divisions in many systems and therefore the regulation of asymmetric and symmetric fates must be tightly regulated for normal development and tissue homeostasis. The lateral "seam" cells in C. elegans are multipotent epidermal stem-like cells aligned along each side of the worm body which produce seam cells, hypodermal cells and neuroblasts. Seam cells undergo an invariant developmental pattern of division, with a single asymmetric self-renewal division at each larval stage, and specifically in the L2 stage, a single additional round of symmetric proliferative division. Heterochronic genes determine the relative timing of developmental events in C. elegans larvae. Mutants of heterochronic pathway display improper transitions between asymmetric and symmetric seam cell divisions, suggesting a role for this pathway in the temporal regulation of asymmetric and symmetric divisions. On the other hand, the Wnt pathway is implicated in cell polarity, self-renewal, somatic cell reprogramming, and tumorigenesis. A noncanonical Wnt pathway, known as "Wnt/b-catenin asymmetry" pathway regulates most of asymmetric cell divisions and polarity in C. elegans cell lineages including seam cells. Here, we report that the reduction of POP-1 (TCF) or LIT-1 (Nemo Like Kinase) by RNAi changes the asymmetric cell fates to symmetric cell fates, generating either two seam cells (in the case of pop-1(RNAi)) or two differentiating cells (in the case of lit-1(RNAi)). The nuclear level of POP-1 is asymmetrically distributed between the seam cell daughters and can be modulated negatively by LIT-1. Therefore LIT-1 has a crucial role in the transmission of Wnt-TCF signals to the asymmetric activity of POP-1. We examined whether the heterochronic genes may interact functionally with POP-1/LIT-1 in the control of asymmetric vs. symmetric seam cell fates. We found that high LIN-14 activity promotes asymmetric seam cell division, perhaps by potentiating the cell asymmetry activity of POP-1/LIT-1, whereas LIN-28 activity seems to promote symmetric seam cell division, perhaps by blunting the asymmetry activity of POP-1/LIT-1. We propose the heterochronic pathway may modulate thresholds for POP-1/LIT-1 activity in the stage-specific transitions between asymmetric to symmetric seam cell fates.