(2019) Development "The people behind the papers - Sungwook Choi."

[Development, 2019]

The control of timing in development is crucial, both within and between tissues. Heterochrony involves shifts in the rate of development of some tissues relative to others, and although the first heterochronic genes were identified in <i>Caenorhabditis elegans</i> in the early 1980s, their role in inter-tissue developmental coordination is still not completely understood. A new paper in Development tackles this problem with an analysis of the role of <i>lin-28</i>, a key heterochronic gene, in worm fertility. We caught up with Sungwook Choi, first author and recently graduated PhD student in Victor Ambros' lab at the University of Massachusetts Medical School in Worcester, to find out more.

Duan, Ye et al. (2020) MicroPubl Biol

Ambros, Victor, Sun, Yongming, Duan, Ye

[MicroPubl Biol, 2020]

RNA-seq is widely used for the quantitative analysis of transcriptomes in the context of studies of gene expression and regulation (Mortazavi et al., 2008; Ozsolak and Milos, 2011; Wang et al., 2009). Generally, RNA-seq protocols employ poly(A) selection for mRNA enrichment. However, poly(A) based enrichment is subject to potential bias depending on the poly(A) status of various mRNAs, which could be particularly undesirable in the context of studying post-transcriptional gene regulatory mechanisms, such miRNA repression (Wu et al., 2006). Therefore, ribosomal RNA (rRNA) depletion is a desirable alternative strategy to enrich for mRNA sequences in RNA-seq sample preparation (Zhao et al., 2014). However, currently available rRNA depletion toolkits were designed for either mammals or bacteria, and hence do not offer an efficient option for rRNA depletion of RNA samples from certain experimental organisms, such as C. elegans.

Duan, Ye et al. MicroPubl Biol

Duan, Ye, Choi, Sungwook, Ambros, Victor, Nelson, Charles

[MicroPubl Biol, 2020]

The application of CRISPR/Cas9 genome editing has revolutionized the genetic analysis of gene function in vivo by enabling highly efficient and precise in loco mutagenesis of defined genomic loci (Adli, 2018). In particular, CRISPR/Cas9 mediated homologous directed recombination (HDR) permits the introduction of complex genetic modifications at single nucleotide resolution (Cong et al., 2013; Dickinson et al., 2013). However, the targeted mutagenesis of essential genes using CRISPR/Cas9 can be challenging since the recovery of deleterious mutations of an essential gene may require a genetic balancer with a wild type copy of the gene (or alternatively a genetic suppressor locus) that can be later outcrossed for phenotypic analysis of novel targeted mutations. Genetic balancers are commonly available for model organisms such as worms or flies, but in the context of CRISPR/Cas9 genome editing, the balancer itself can be edited along with the targeted genomic locus, dramatically reduce the efficiency of recovering the desired loss of function alleles. Therefore, editing of essential genes would be facilitated by a strategy where the targeted genomic locus is efficiently edited, whilst the balancer locus is resistant to editing.

Winkenbach LP et al. (2019) RNA Biol "Todos Santos small RNA symposium."

Claycomb JM, Doser R, Pasquinelli AE, Winkenbach LP, Reed KJ, Phillips CM

[RNA Biol, 2019]

Worm biologists from the United States, Canada, and the United Kingdom gathered at the Colorado State University Todos Santos Center in Baja California Sur, Mexico, April 3-5, 2019 for the Todos Santos Small RNA Symposium. Meeting participants, many of whom were still recovering from the bomb cyclone that struck a large swath of North America just days earlier, were greeted by the warmth and sunshine that is nearly ubiquitous in the sleepy seaside town of Todos Santos. With only 24 speakers, the meeting had the sort of laid-back vibe you might expect amongst the palm trees and ocean breeze of the Pacific coast of Mexico. The meeting started with tracing the laboratory lineages of participants. Not surprisingly, the most common parental lineages represented at the meeting were Dr. Craig Mello, Dr. Gary Ruvkun, and Dr. Victor Ambros, whom, together with Dr. Andy Fire and Dr. David Baulcombe, pioneered the small RNA field. In sad irony, on the closing day of the meeting, participants were met with the news of Dr. Sydney Brenner's passing. By establishing the worm, <i>Caenorhabditis elegans</i>, as a model system Dr. Brenner paved the way for much of the research discussed here.

Cale, Allison R et al. (2020) MicroPubl Biol

Karp, Xantha, Cale, Allison R

[MicroPubl Biol, 2020]

In response to adverse environments C. elegans larvae may form stress-resistant and developmentally arrested dauer larvae. Entry into dauer interrupts developmental progression after the second larval molt (Cassada and Russell 1975), and the dauer formation decision must be coordinated with developmental pathways. Heterochronic genes control stage-specific cell fate decisions, and these genes interconnect with the dauer formation (daf) pathway in several ways. Dauer formation and daf genes affect heterochronic phenotypes, and conversely, heterochronic genes can impact dauer by controlling the timing of dauer formation, the morphology of dauer larvae, or the ability to enter dauer (Liu and Ambros 1989; Liu and Ambros 1991; Antebi et al. 1998; Tennessen et al. 2010; Karp and Ambros 2011; Karp and Ambros 2012). Here we find that the lin-41 heterochronic gene regulates the dauer formation decision and dauer morphogenesis via its canonical target, lin-29. The temperature-sensitive daf-7(e1372) allele results in constituitive dauer entry at 25&#730;C, and the allele is a useful tool for controlling dauer entry (Vowels and Thomas 1992; Karp 2018). While performing experiments examining the role of lin-41 during dauer, we observed that lin-41(0); daf-7 worms rarely entered dauer. To better understand this phenotype, we quantified the number of dauer and nondauer larvae in synchronous populations of daf-7(e1372) larvae grown at the dauer-inducing temperature of 24&#730;C.lin-41(0) mutants are sterile, and therefore the strain must be propagated as a heterozygote (Slack et al. 2000). We took advantage of a closely linked transgene, nIs408[LIN-29::mCherry, ttx-3::gfp] to balance lin-41(0) (Harris and Horvitz 2011). lin-41(0) homozygous larvae were identified by the absence of fluorescence. Controls included nIs408-positive siblings, 2/3 of which should be lin-41(0)/nIs408 and 1/3 of which should be nIs408/nIs408, as well as a strain that is homozygous for the wild-type allele of lin-41.

Pasquinelli AE (2002) Trends Genet "MicroRNAs: deviants no longer."

Pasquinelli AE

[Trends Genet, 2002]

Almost ten years ago, the Ambros laboratory made the extraordinary discovery that a gene essential for development in Caenorhabditis elegans encoded a 22-nucleotide, untranslated RNA. Further genetic studies in this nematode revealed the existence of a second tiny RNA gene that turned out to be conserved in animals as diverse as flies and humans. Now, the Ambros, Bartel and Tuschl laboratories have proven that those odd RNAs were just the first examples of a large family of RNAs, termed microRNAs (miRNAs). Although untranslated RNA genes, such as transfer RNAs and ribosomal RNAs, perform essential housekeeping roles in all living organisms, growing numbers of other RNAs, some widely conserved across phyla and others limited to certain species, are being uncovered and shown to fulfill specific duties. The discovery of miRNAs establishes a new class of regulatory RNAs and highlights the existence of unexpected RNA genes that, although ancient, are not extinct.

Robins H et al. (2005) Proc Natl Acad Sci U S A. "Incorporating structure to predict microRNA targets."

Li Y, Robins H, Padgett RW

[Proc Natl Acad Sci U S A., 2005]

MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including Caenorhabditis elegans, Drosophila, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. M Tuschl, T. (2001) Science 294, 853-858; Lai, E. C., Tomancak, P., Williams, R. W. M Rubin, G.M. (2003) Genome Biol. 4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. M Bartel, D. P. (2001) Science 294, 858-862; Lee, R. C. M Ambros, V. (2001) Science 294, 862-8644; and Lee, R. C., Feinbaum, R. L. M Ambros, V. (1993) Cell 115, 787-798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3' UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using Drosophila miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is mad as a target for bantam. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported.

Yang, Bing et al. MicroPubl Biol

Yang, Bing, McJunkin, Katherine

[MicroPubl Biol, 2020]

The mir-35-42 family of microRNAs (miRNAs) acts redundantly to ensure embryonic viability in C. elegans (Alvarez-Saavedra and Horvitz 2010). We are interested in defining the essential targets that must be repressed by the mir-35-42 family. Our previous work suggested that NHL (ring finger b-box coiled coil) domain containing 2 (nhl-2) may be one such target because genome editing attempts to delete the mir-35-42 seed binding region in the nhl-2 3UTR were unsuccessful (McJunkin and Ambros 2017). The same CRISPR reagents were successful at creating such a deletion in a background containing an NHL-2 CDS deletion (nhl-2(ok818)) (McJunkin and Ambros 2017). Together, we took these results to mean that derepression of nhl-2 induced lethality or sterility, preventing our isolation of the deletion lines in the wild type context. More recently, CRISPR genome editing reagents and protocols have become many-fold more efficient, most notably by injection of recombinant Cas9 RNPs pre-loaded with synthetic guide RNAs (gRNAs) (Paix et al. 2014). Using injection of Cas9/gRNA RNPs, we have succeeded in deleting and mutating the mir-35-42 seed binding region in the nhl-2 3UTR in a wild type background (see alleles in Figure 1A). Because such alleles were previously difficult to generate, we quantified their fecundity and embryonic viability (which are two aspects of physiology affected by mir-35 family mutations) (Alvarez-Saavedra and Horvitz 2010; McJunkin and Ambros 2014) to see if they were impaired, but we found these animals to be wild type (Figure 1B). Therefore, our original interpretation that the difference in CRISPR editing between wild type and nhl-2(ok818) backgrounds was due to negative selection of miRNA binding site mutations in the wild type background was incorrect. One possible explanation for the observed difference in editing may be alterations in chromatin structure induced by the 1.5kb nhl-2(ok818) deletion. Indeed, nucleosome position and dynamics have been shown to alter efficiency of Cas9 cleavage (Chen et al. 2016; Horlbeck et al. 2016; Isaac et al. 2016; Hinz et al. 2016; Daer et al. 2017; Yarrington et al. 2018; Kim and Kim 2018). Thus, differences in genome editing efficiencies between genetic backgrounds should be interpreted with caution.

Manikandan J et al. (2008) Bioinformation "Oncomirs: the potential role of non-coding microRNAs in understanding cancer."

Aarthi JJ, Manikandan J, Pushparaj PN, Kumar SD

[Bioinformation, 2008]

MicroRNAs (miRNAs) are members of a family of non-coding RNAs of 8-24 nucleotide RNA molecules that regulate target mRNAs. The first miRNAs, lin-4 and let-7, were first discovered in the year 1993 by Ambros, Ruvkun, and co-workers while studying development in Caenorhabditis elegans. miRNAs can play vital functions form C. elegans to higher vertebrates by typical Watson-Crick base pairing to specific mRNAs to regulate the expression of a specific gene. It has been well established that multicellular eukaryotes utilize miRNAs to regulate many biological processes such as embryonic development, proliferation, differentiation, and cell death. Recent studies have shown that miRNAs may provide new insight in cancer research. A recent study demonstrated that more than 50% of miRNA genes are located in fragile sites and cancer-associated genomic regions, suggesting that miRNAs may play a more important role in the pathogenesis of human cancers. Exploiting the emerging knowledge of miRNAs for the development of new human therapeutic applications will be important. Recent studies suggest that miRNA expression profiling can be correlated with disease pathogenesis and prognosis, and may ultimately be useful in the management of human cancer. In this review, we focus on how miRNAs regulate tumorigenesis by acting as oncogenes and anti-oncogenes in higher eukaryotes.

Nigon VM et al. (2017) WormBook "History of research on C. elegans and other free-living nematodes as model organisms."

Nigon VM, Felix MA

[WormBook, 2017]

The nematode Caenorhabditis elegans is now a major model organism in biology. The choice of Sydney Brenner to adopt this species in the mid-1960s and the success of his team in raising it to a model organism status have been told (http://www.wormbook.org/toc_wormhistory.html; Brenner, 2001; Ankeny, 2001). Here we review the pre-Brenner history of the use of free-living nematodes as models for general questions in biology. We focus on the period that started in 1899 with the first publication of Emile Maupas mentioning Rhabditis elegans and ended in 1974 with the first publications by Brenner. A common thread in this period, aided by the variety in modes of reproduction of different nematode species, is found in studies of meiosis, fertilization, heredity and sex determination. Maupas in his 1900 opus on reproduction already chooses C. elegans as the species of reference. Hikokura Honda determined its hermaphrodite chromosomal content in 1925. C. elegans was again isolated and chosen as main subject by Victor Nigon in the 1940-50s. Nigon mastered crosses between C. elegans hermaphrodites and males, described the meiotic behavior of chromosomes in XX hermaphrodites and X0 males and, using tetraploids, correctly inferred that sex was determined by X chromosome to autosome dosage. With Ellsworth Dougherty, Nigon isolated and studied a C. briggsae body size mutant and a C. elegans slow growth mutant. Dougherty and his team devoted most of their work to finding a defined culture medium to screen for physiological mutants, focusing on C. briggsae. With Helene Fatt, Dougherty also performed the first genetic study of natural variation in C. elegans, concerning the difference in heat resistance of the Bergerac and Bristol strains. Jean Brun, a student of Nigon, performed a long and remarkable experiment in acclimatization of C. elegans Bergerac to higher temperatures, the significance of which remains to be clarified.