Figure 1.
Genetic network of heterochronic genes regulating the developmental timing of cell divisions associated with the larval stages of C. elegans development.
Based on [1].
Figure 2.
Four of five newly isolated alg-1 alleles carry missense mutations.
(A) A schematic showing the positions of the newly identified alg-1 mutations within the ALG-1 protein (black tics) and the existing deletion alleles (red lines). Positions of catalytic sites are indicated by yellow circles. (B) Exon/intron schematic of the two alg-1 isoforms predicted and supported by cDNA evidence (Wormbase.org). Boxes represent exonic regions. (C). Western blot analysis on total protein lysate from wild type and alg-1 mutant animals. All non-null alg-1 alleles, like the wild type, produce 2 isoforms of ALG-1. Newly identified missense alleles of alg-1 are marked in red and null alleles are in blue. alg-1(tm369) is a loss of function allele that deletes most of the PIWI domain and produces 2 truncated isoforms of ALG-1(*). All strains with the exception of alg-1(tm492) and alg-1(tm369) carry lin-31(lf) and col-19::gfp in the background.
Table 1.
alg-1 mutations cause retarded development.
Figure 3.
Mutations in alg-1 suppress precocious development of lin-28(lf) (lin-28(n947)) animals.
(A, B) alg-1 mutations suppress the egg-laying defect of lin-28(lf) animals by suppressing the precocious divisions of the vulval precursor cells; (B) Early third larval (eL3) stage animals. Arrowheads indicate vulval cell nuclei. Three vulval precursor cells (P5.p, P6.p, and P7.p) are undivided in the top (N2) and bottom (lin-28(n947); alg-1(ma192)) panels, but in the middle panel (lin-28(n947), P6.p and P7.p have divided twice (one P7.p granddaughter is out of the plane of focus). (C, D) alg-1 mutations suppress the lin-28(lf) precocious expression of the adult cell fate marker col-19::gfp. lL4-late fourth larval stage. (E) alg-1 mutations also increase the seam cell number (#) produced lin-28(lf) mutant animals (***p<0.0001), and (F) suppress precocious alae formation of L4 animals; dotted line represents absence of the alae, solid line underlines the alae structure. All strains col-19::gfp transgene in the background. n = number of animals scored.
Table 2.
lin-28(lf) phenotypes are suppressed by mutations in alg-1.
Figure 4.
Newly isolated alg-1 alleles are antimorphic, exhibit retarded development, and display phenotypes more severe than those of alg-1(0).
(A, B) Bar graphs showing the percent of young adult (YA) animals with wild type alae formation (A) and col-19::gfp adult marker expression (B), where alg-1(anti) alleles have a dosage dependent effect on both phenotypes. (C) Schematic of representative V1–V4 and V6 lineage cell divisions in the wild type, alg-1(anti), and other heterochronic mutants. (D) alg-1(anti) mutations display increased numbers of seam cells as young adults. ***p<0.001. All strains carry lin-31(lf) and col-19::gfp in the background. The lin-31 mutation is present in order to suppress alg-1(anti) vulval bursting phenotypes by non-heterochronic methods. n = number of animals scored.
Figure 5.
alg-1(anti) mutations affect hbl-1 expression.
(A) hbl-1::gfp::hbl-1 3′UTR expression in wild type and alg-1(anti) mutant animals. mL3-mid third larval stage. Short arrows-hypodermal nuclei; long arrows-seam cell nuclei. HSN neuron (arrowhead) is shown as a point of reference. Images were captured at identical exposure, and processed identically. (B) Quantification of hypodermal hbl-1::gfp::hbl-1 3′UTR expression in wild type and alg-1(ma202) animals. (C) hbl-1 RNAi rescues the retarded expression of the adult marker col-19::gfp in alg-1(anti) mutant animals. All strains carry lin-31(lf) and col-19::gfp in the background. The lin-31 mutation is present in order to suppress alg-1(anti) vulval bursting phenotypes by non-heterochronic methods. n = number of animals scored.
Figure 6.
alg-1(anti) mutations affect functions of many microRNAs and exhibit phenotypes more severe than those of alg-1(0).
(A) Plim-6::gfp expression marks ASEL neuronal cell fate in wild type and mutant animals. lsy-6(ot150) mutants lack the Plim-6::gfp expression in the ASEL neurons some of the time. This phenotype is enhanced by the loss of alg-1 and even more so in the presence of either of the two alg-1(anti) mutations. All strains carry lin-31 mutation in order to suppress alg-1(anti) vulval bursting phenotypes by non-heterochronic methods. (B) Combination of alg-1(anti) and alg-2(0) mutations results in embryonic lethality (emb) and early larval lethality (let), with some late larval lethality present in alg-2(0)/+; alg-1(anti)/alg-1(anti) mutant animals. (C, D) Distal tip cell migration phenotypes of wild type and mutant animals. alg-1(anti) mutant animals display defects affecting all three phases of gonad migration. In (C) gonads are marked by a dashed line. (E) alg-1(anti), but not alg-1(0) mutation enhances the embryonic lethality of mir-35–41 mutants. (**p<0.001). All strains carry lin-31(lf) and col-19::gfp in the background. The lin-31 mutation is present in all strains in order to suppress alg-1(anti) vulval bursting phenotypes by non-heterochronic methods. n = number of animals scored.
Figure 7.
The effect of alg-1 mutation on the levels of mature microRNAs in total RNA from L2 larvae using FirePlex miRSelect assay.
(A, B) Quantification of let-7-Family microRNA abundance (normalized to wild type), in the context of suppression of lin-28(0) mutants by alg-1 mutations (A), and for alg-1 mutations in the lin-28(+) genetic background (B). Only detectable let-7-Family microRNAs are shown. (A) let-7 microRNA levels increase dramatically in lin-28(lf) mutants and are reduced by the addition of the alg-1 mutations. Levels of other let-7 family members are not increased to statistically significant levels in lin-28 mutants, and are either not affected or are decreased by the alg-1 mutations to varying degrees. All strains carry the col-19::gfp transgene. (B) let-7-Family microRNA levels are decreased in all alg-1 mutants compared to wild type, but alg-1(0) decreases microRNA abundance more than alg-1(anti) mutations do. (C, D) Scatterplots comparing abundance of microRNAs in total RNA from L2 larvae of wild type (X-axis, arbitrary units) and alg-1 mutants (Y-axis, arbitrary units) using FirePlex miRSelect assays for 53 microRNAs. Complete loss of ALG-1 in alg-1(0) and compromised ALG-1 function in alg-1(ma202) and alg-1(ma192) results in under accumulation of microRNAs. alg-1(ma202) and alg-1(ma192) mutants have higher levels of microRNAs than alg-1(0) animals, *p = 0.01, **p = 0.003. (D) Subset of data in (C) zoomed in to show the lower abundance microRNAs. All strains in (B–D) carry lin-31(lf) and col-19::gfp in the background. The lin-31 mutation is present in order to suppress alg-1(anti) vulval bursting phenotypes by non-heterochronic methods.
Figure 8.
alg-1(anti) alleles affect conserved amino acids within the ALG-1 protein, do not affect pre-microRNA processing, and associate with microRNAs to levels comparable to wild type.
(A) Alignments of Argonaute sequences from multiple species shows conservation of the amino acids affected by alg-1(anti) mutations. (B) Locations of conserved amino acids affected by alg-1(anti) mutations are mapped onto the crystal structure of hAGO2 (PDB ID 4EI1 [51]) using PYMOL. PAZ domain colored in blue, Linker 2 in yellow, MID domain in green, and PIWI domain in red. Amino acids affected by each mutation are highlighted in teal. (C) A schematic showing precursor microRNA processing into the mature guide microRNA. (D) Northern blot analysis of total RNA extracted from mixed population wild type and alg-1 mutant animals using probes to specific microRNAs. Only alg-1(0) null and alg-1(tm369) loss of function mutants accumulate the precursor species of microRNAs. *Strains carry lin-31, col-19::gfp in the background. m/SL2, ratio of mature microRNA to SL2 loading control normalized to wild type lin-31; col-19::gfp. pre/m, ratio of precursor to mature microRNA normalized to wild type lin-31; col-19::gfp. (E) Scatterplot comparing the efficiency with which microRNAs co- immunoprecipitated wild type (X-axis) or mutant (Y-axis) ALG-1. microRNA extracted from ALG-1 immunoprecipitations was quantified using Taqman qRT-PCR. microRNA abundance in each IP was normalized to a synthetic spike-in, and also to the amount of microRNA in the starting material and to the amount of ALG-1 immunoprecipitated (IP-ed). The graph shows average % IP-ed from 3 biological replicates. (F) Subset of data in (E), showing % of let-7-Family microRNAs IP-ed with ALG-1. All strains in (E, F) carry lin-31(lf) and col-19::gfp in the background. The lin-31 mutation is present in order to (non-heterochronically) suppress alg-1(anti) vulval bursting phenotypes.
Figure 9.
Western blot analysis of ALG-1 immunoprecipitated complexes from extracts of alg-1(anti) and wild type animals.
Immunoprecipitated ALG-1(anti) shows an increased association with DCR-1, and a decreased association with AIN-1, compared to wild type ALG-1. The ratio of DCR-1 to ALG-1 and AIN-1 to ALG-1 were determined by quantitation of the Western blot signals, and each of those ratios for the alg-1(anti) mutants is normalized to that of the wild type. * means not applicable (n/a).
Figure 10.
Purified recombinant ALG-1(anti) retain the slicing ability similar to the wild type ALG-1.
(A) SDS-PAGE gel of recombinant ALG-1 proteins. Wild type ALG-1, ALG-1 G553R and ALG-1 S895F were bacterially expressed and purified. (B) Radiolabeled target RNA is cleaved by rALG-1 proteins preloaded with a siRNA (red) to produce a major 15 nt cleavage species as well as two minor cleavage species. (C) rALG-1 proteins, including rALG-1(anti), bind and cleave a perfectly base-paired duplex. (D) rALG-1 proteins bind a microRNA duplex containing containing two mismatches. Pre-bound rALG-1/duplex complexes are able to cleave an RNA target, producing a 15-nt major cleavage RNA species.
Figure 11.
Model representing a proposed mode of action of antimorphic mutant ALG-1.
(A) In wild type animals, ALG-1 and ALG-2 associate with microRNA precursors (Loading), act in conjunction with Dicer to facilitate cleavage of the precursor to yield mature microRNA (Dicing), and through a series of miRISC maturation steps, associate with effector partners including AIN-1/GW182, and repress mRNA targets (Maturation/Function). According to the model, ALG-1 and ALG-2 are partially redundant, and function asymmetrically; ALG-1 carries a majority share of microRNA function. (B) In the absence of ALG-1, ALG-2 is able to partially cover for reduced overall ALG activity, and so alg-1(0) animals exhibit weak microRNA loss-of-function phenotypes, and accumulate abnormal levels of microRNA precursors. (C) In alg-1(anti) animals, microRNA activity is globally poisoned by the ALG-1(anti) protein. This is because ALG-1(anti) protein is competent for Loading and Dicing, but is blocked in one or more steps of miRISC maturation wherein ALG-1 would normally transition from Dicer associated microRNA processing to AIN-1/GW182 associated target repression. This hypothetical miRISC maturation defect results in sequestration of miRISC components (including microRNAs) in inactive complexes. Jagged lines attached to ribosomes (brown ovals) represent un-repressed protein synthesis from target mRNA.