Paper for the discussion :
Parry DH, Xu J, Ruvkun G. A whole-genome RNAi Screen for C. elegans miRNA pathway genes. Curr Biol. 2007 Dec 4;17(23):2013-22.
You will need Figure 1A from supplemental data.
Questions for the discussion:
1)What are the major advantages and disadvantages of RNAi screens over classical genetic screens and, in particular, miRNA Pathway Genes?
- recover loss-of-function mutations;
- recover gain-of function mutations;
- provide insights into structure-function relationships;
- affect single gene;
- uncover redundancy;
- every gene can be targeted;
- bypass earlier requirements;
- the cloning stage is eliminated to identify the targeted gene;
- heritable.
2)What is the role of heterochronic genes, especially the let-7 gene, in C. elegans development?
3)Why was it necessary to use the eri-1(mg366);let(mg279) mutant background for this particular screen? (Fig. S1A)
Describe the screen briefly.
4)What was the purpose of the following genetic tests?
- eri-1(mg366);let(mg279) vs eri-1(mg366);
- eri-1(mg366);let(mg279) vs lin-41(ma104); eri-1(mg366);let(mg279);
5)What other heterochronic phenotypes were induced by gene inactivation that are consistent with defects in let-7 miRNA function?
6)What kind of assays were perfomed to reveal if these genes were involved in let-7 biogenesis?
7)Where does a major subset of these genes function in the miRNA pathway?
8)What are the groups of genes identified by this screen?
RNAi feeding in Caenorhabditis elegans
To carry out RNAi by feeding in Caenorhabditis elegans, a bacterial strain that
expresses double-stranded (ds) RNA corresponding to sequence of an individual gene is generated and fed to worms. Typically, dsRNAs are 200–2,000 bp long.
Two RNAi feeding libraries are currently available:
-expressing ds RNA corresponding to genomic DNA fragments (C. elegans genes have on everage very small introns);
-expressing ds RNA corresponding to ORFs.
Feeding is carried out:
-in 96-well liquid culture (rapid using liquid-handling devices and idial for assays such as life vs death)
-on lawns of bacteria on agar plates (allow more detailed phenotypic scoring, for example, of morphological defects.
C. elegans can be subjected to RNAi at any stage and assayed later, or mothers can be treated and their progeny scored. The latter method is used for embryonic phenotypes to circumvent maternal effects and impermeability of eggshell to dsRNA.
RNAi-supersensitive strains are available and should be considered. These are especially useful for screens involving the nervous system, in which RNAi by feeding is less effectiveeffective.
Adopted from: Boutros M, Ahringer J. The art and design of genetic screens: RNA interference. Nat Rev Genet. 2008 Jul;9(7):554-66.
En example of heterochronic gene activities in lateral hypodermis (lin-4, lin-14, let-7, lin-41) .
Larva 1
H, V and T lineages produce lateral hypodermis.
P3-P8 produce hypodermal cells of vulva.
At hatching, there are two rows of 10 seam cells on each side of the animal (H0 –T) which are embedded in hyp7 and are in close contact with the ventral epithelial (P) cells. At the beginning of each larval stage the seam cells H1, H2, V1-6 and T undergo cell division whereas the most anterior cell, H0, does not divide. As a general rule the posterior daughter of each seam cell division becomes a seam cell while the anterior daughters become detached in their apical surfaces and fuse to hyp 7 syncytium. At the same time, each newly born seam cell elongates and reaches its neighbor on both sides to make another row. This pattern of division, hyp 7 fusion, and cell elongation repeats itself in each of the larval stages until mid-L4. At mid-L4, 15 newly born seam cells in each row elongate longitudinally and fuse with each other and H0 to form two continuous lateral syncytia of seam cells (with 16 nuclei each) mostly embedded in the hyp 7 syncytium.
Adopted from: ATLAS OF C. elegans ANATOMY. Chapter 2. Adult organs and tissues 1. Epithelial System. Hypodermis.
As an example, a lineage defect in the seam cells associated with lin-4, let-7 and lin-14, lin-41 loss-of-function mutants are shown below. Seam cell terminal differentiation is represented by 3 horizontal bars.
wild type - normal
lin-4 (e912) – retarded
let-7 (n2853) – retarded
lin-41 (n2914) – precocious
lin-14 (0) -precocios
LIN-14 expression level is decreased by lin-4 RNA expression at the end of the 1rst larval stage to allow progression to late larval stages. In late larval stages, the expression of LIN-41 and other genes is similarly downregulated by the let-7 RNA, relieving their repression of LIN-29 protein expression and allowing progression to the adult stage.
Vella, M.C. and Slack, F.J. C. elegans microRNAs (September 21, 2005),
WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/
wormbook.1.26.1,
let-7
The let-7 miRNA acts in the heterochronic pathway to control the timing of developmental events at the L4 to adult transition.
Strong loss of function alleles of let-7 cause bursting at the vulva, whereas the weak allele, mg279, reduces the level of mature let-7, but does not cause bursting.
let-7(mn112) null - deletion of transcription start site and upstream region;
let-7(n2853) strong lf -alter the 5th nucleotide of the let-7 transcript.
let-7(mg279) weak lf – deletion of a possible transcriptional regulatory domain upstream
of the let-7 transcript.
Busting vulva phenotype in let-7 mutant.
The retarded alae phenotype in let-7 mutant.
The 1st evidence of a let-7 heterochronic defect is at the L4-to adult moult. Hypodermal blast cells normally divide at each larval stage and at the adult stage exit the cell cycle, fuse with neighboring hypodermal seam cells and generate cuticular alae. In let-7(n2853) animals, the blast cell lineages were normal through the L3-to-L4 moult, but at the L4-to-adult moult, they reiterated larval patterns of cell division and failed to generate alae.
Adopted from: B.J. Reinhart et al. Nature 403 (2000), pp. 901–906.
Page A.P. and Johnstone, I.L. The cuticle (March 19, 2007), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/ wormbook.1.138.1,
C. elegans exoskeleton collagen COL-19 is an adult-specific.
Collagens represent the major structural extracellular matrix (ECM) proteins. C. elegans forms two major collagenous ECMs, the cuticular exoskeleton and the tissue-surrounding basement membranes. The cuticle protects the worm from the environment, prevent desiccation, and acts as a hydrostat. The nematode cuticle collagens are synthesized and secreted from the underlying hypodermis.
Col-19 is an adult-specific, hypodermally synthesized collagen, localized to the circumferential annular rings and the lateral trilaminar alae of the cuticle. This collagen is synthesized by the syncytial ventral and dorsal hypodermal cells that cover the head, tail, ventral and dorsal surfaces, and the distinct lateral seam cells that terminally differentiate to form two parallel syncytial bands or cords at the fourth larval to adult molt.
From Thein et al. 2003. Dev. Dynamics. 226:523–539.
lin-41
Lin-41 is a direct target of let-7 (contains two let-7 binding sites in 3’UTR). The retarded alae phenotype and busting vulva phenotype caused by let-7 mutations can be suppressed by precocious mutations in the genes lin-41,lin-41 is negatively regulated by let-7.
From: B.J. Reinhart et al. Nature 403 (2000), pp. 901–906.
eri-1
C. elegans eri-1 was identified in a genetic screen for mutants with enhanced sensitivity to dsRNAs (Kennedy S. et al. 2004. Nature, 427: 645). eri-1encodes an evolutionarily conserved protein with domains homologous to nucleic-acid-binding and exonuclease proteins. ERI-1 is a negative regulator that may normally function to limit the duration, cell-type specificity or endogenous functions of RNA interference. After exposure to dsRNA or siRNAs, animals with eri-1 mutations accumulate more siRNAs than do wild-type animals. C. elegans ERI-1 and its human orthologue degrade siRNAs in vitro. ERI-1 is predominantly cytoplasmic and is expressed most highly in the gonad and a subset of neurons, suggesting that ERI-1 siRNase activity suppresses RNA interference more intensely in these tissues.