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Comparative Study
. 2005 Nov 29;102(48):17360-5.
doi: 10.1073/pnas.0508989102. Epub 2005 Nov 15.

Negative regulation of nuclear divisions in Caenorhabditis elegans by retinoblastoma and RNA interference-related genes

Alla Grishok  1 , Phillip A Sharp
Affiliations
Comparative Study

Negative regulation of nuclear divisions in Caenorhabditis elegans by retinoblastoma and RNA interference-related genes

Alla Grishok et al. Proc Natl Acad Sci U S A. .

Abstract

Short RNA regulatory molecules, microRNAs, and short interfering RNAs participate in a range of developmental gene networks by base-pairing with their target sequences. Consistent with these findings, genes required for the biogenesis and function of short interfering RNAs and microRNAs, dicer (dcr-1 in Caenorhabditis elegans) and argonaute homologs, are essential for development in diverse organisms, including C. elegans. We demonstrate that genes required for the function of short RNAs synergize with the retinoblastoma tumor suppressor homolog lin-35 in negative regulation of the nuclear divisions in the intestine of C. elegans. The level of cyclin E (cye-1) expression is critical for nuclear divisions in the intestine and is elevated in double mutants in lin-35 and RNA interference pathway genes. We propose that RNA interference-related pathways cooperate with retinoblastoma in transcriptional repression of endogenous genes, an example being cyclin E.

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Figures

Fig. 1.
Fig. 1.
dcr-1 and alg-1/2 negatively regulate nuclear divisions in the intestine. (A) The elt-2::gfp reporter strain shows 30 intestinal nuclei. (B) The number of intestinal nuclei increases in alg-1/2(RNAi) animals because of extra divisions of some nuclei (bracket). (C) The number of intestinal nuclei increases dramatically in lin-35(n745);alg-1/2(RNAi) animals. For A–C ×10 lens magnification was used. (D) Quantification of postembryonic nuclear divisions in the intestine (number of nuclei in adult worms after subtraction of 20 nuclei present in L1) in different genetic backgrounds. For all figures, intestinal nuclei were counted in 15–30 progeny of several worms subjected to RNAi by feeding, and data for each genotype are presented as a mean number ± SD. RNAi feeding experiments were repeated at least three times.
Fig. 2.
Fig. 2.
miRNA and RNAi pathway genes synergize with lin-35(Rb). (A) Dissected intestines of WT (Upper) and alg-1(gk214);lin-35(n745) double mutant (Lower) adult worms were stained with DAPI. The bracket in Lower highlights the increased number of nuclei. A ×10 lens magnification was used, and exposure times were identical. (B and D) Quantification of postembryonic intestinal nuclear divisions in different genetic backgrounds with elt-2::gfp transgenic strains; alg-1(Z) and lin-35(Z) indicate that only the zygotic complement of the gene product was missing, whereas the maternal contribution was still present. (C) A differential interference contrast microscopy image of the midsection of the representative rde-4(ne299);lin-35(n745) mutant worm at ×40 lens magnification. Ventral is top; double vulva protrusions are shown by arrows, and abnormal gonad migration with two U-turns is indicated by the white line. Note the accumulation of the late-stage embryos (*) caused by egg-laying defects.
Fig. 3.
Fig. 3.
The level of cyclin E is critical for nuclear divisions in the intestine and is affected by lin-35 and RNAi pathways. (A) Quantification of postembryonic intestinal divisions in WT, cye-1(eh10) heterozygous, and homozygous animals in the absence (Left) and presence (Right) of alg-1/2(RNAi). (B) Expression of cye-1::gfp (KM32) reporter in the anterior intestine of adult WT type (Upper) or alg-1/2(RNAi);lin-35(RNAi)(Lower) animals. (C) Expression of cye-1::gfp reporter in the hypodermal seam cells (arrowheads) of adult WT worms (Top) or worms treated with RNAi against the indicated genes. Exposure times in all images in B and C were identical.
Fig. 4.
Fig. 4.
Molecular analysis of the regulation of cye-1 expression suggests a direct role for the RNAi pathway. (A) Real-time RT-PCR analysis of the expression levels of cye-1 mRNA in different mutant backgrounds. Levels of cye-1 mRNA were normalized to ama-1 mRNA levels. Results of three independent experiments are shown as mean relative numbers ± SD. (B) Quantification of the results of ChIP experiments by real-time PCR with primers 3 and 4 and the indicated antibodies. For each DNA sample PCR with actin-specific primers was used for normalization of the signal. Relative ratios are presented where PCR signal from control DNA samples from WT worms is taken as 1. Sequences for primers used are in Materials and Methods.
Fig. 5.
Fig. 5.
Antisense transcription at the cye-1 locus. (A and B) Schematic representations of the 600-bp region upstream of the SL1 acceptor site in cye-1 gene and locations of the primers used for RT and PCRs are shown at the top. Results of the RT-PCRs detecting antisense transcription at the cye-1 locus are shown at the bottom. The identity of the bands was confirmed by sequencing. Sequences for primers used are in Materials and Methods. (C) Model proposing negative regulation of cyclin E gene expression by parallel Rb and RNAi-TGS pathways.
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