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PNAS 105 (35): 12897-12902

Copyright © 2008 by the National Academy of Sciences.

A starvation-induced noncoding RNA modulates expression of Dicer-regulated genes

Sabine Hellwig Brenda L. Bass*

Department of Biochemistry and Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112


Figure 1
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Fig. 1.. Genomic location, structure, and expression of rncs-1. (A) The X chromosome region that encodes the 847-nt rncs-1 gene is shown. Gray boxes, coding exons; white boxes and arrows, noncoding exons; connecting lines, introns. The 815-bp deletion in rncs-1(tm1632) is indicated. Regions used as promoter in Prncs-1::GFP and as rescue fragment in rncs-1 rescue and overexpressing lines are marked. (B) Secondary structure of mature rncs-1 RNA as predicted by mfold and biochemical methods (nuclease probing; data not shown). Asterisks, GU pairs; dots, mismatches and bulges; gray arrowhead, exon–exon junction; numbering, nucleotide relative to transcription start. (C) Northern blot of rncs-1 in total RNA from wild-type embryos, larval stages (L1–L4), young adults, arrested L1s, and dauer larvae purified from exhausted liquid culture. Blot was rehybridized with probe for 18S ribosomal RNA (rRNA) as a loading control. Data are representative of multiple blots (n = 2–4, depending on stage). (D–F) GFP expression from putative rncs-1 upstream regulatory and promoter sequences. (D) Comma stage embryo with fluorescence in the developing midgut (dmid) and cells of developing hypodermis (dh). (E and F) Adult with Prncs-1::GFP expression in the midgut (mid), cells of the head hypodermis (hh), tail hypodermis (th), Hyp 7 syncytium (Hyp 7), and vulval epithelium (ve). GFP expression is absent in seam cells (sc). Focal planes are shown at Bottom Right. Because transgenes are often silenced in the germ line, the lack of GFP expression in the germ line is not necessarily indicative of lack of expression. Multiple transgenic lines showed the same expression pattern. (G) Northern blot of rncs-1 in total RNA of wild-type hermaphrodites (H) and males (M); 18S rRNA, loading control. (H) Quantification of relative RNA levels in four independent samples of males and hermaphrodites. rncs-1 levels were determined by Northern blotting; let-413 and eps-8 levels were quantified by qRT-PCR. Error bars, SEM; *, P < 0.05, t test.

 

Figure 2
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Fig. 2.. Regulation of rncs-1 transcription in response to food supply. (A) Northern blot for rncs-1 in RNA of wild-type L4 larvae starved for indicated times and then reintroduced to food for several hours (48-h starved, 1- to 6-h fed); 18S rRNA, loading control. (B) Quantified Northern blot data for two independent cohorts of worms. Black diamonds/solid line, rncs-1 RNA; white squares/dashed line, total polyadenylated RNA detected by an oligo(dT) probe; black arrowhead, time of food addition; error bars, SEM. Transcript levels were normalized to 18S rRNA and are shown relative to unstarved 0-h sample. The possibility that the increase in rncs-1 levels compared with rRNA was caused by reduction of total RNA during starvation was excluded by measuring total RNA yield per worm in a starvation time course (Fig. S1). (C) Comparison of rncs-1 levels in well fed L3 larvae (L3), dauer larvae isolated from starved and overcrowded liquid culture (dau), and dauer larvae grown with food on dauer pheromone-rich plates (dau-ph). (D) Northern blot of GFP mRNA and rncs-1 in RNA from wild-type worms carrying the Prncs-1::GFP transgene that were subjected to a starvation/feeding time course; 18S rRNA, loading control.

 

Figure 3
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Fig. 3.. rncs-1 is not cleaved by Dicer but inhibits Dicer activity in vitro. (A) Schematic of full-length rncs-1 (1) and derivatives (2–5) synthesized by in vitro transcription. Substrate 5 is identical to 4 except GU base pairs and mismatches were repaired. (B) Autoradiogram showing reaction products of 1-h incubations of 32P-labeled rncs-1 and derivatives in wild-type embryo extract as analyzed by denaturing gel electrophoresis. Processing of dsRNA (ds) by Dicer is evidenced by the appearance of siRNA (si). Size standards, RNA Decade Markers (Ambion). (C) Inhibition of Dicer cleavage of a 32P-labeled 300-bp dsRNA (20 nM; unc-22; see Materials and Methods) by unlabeled rncs-1. Autoradiogram shows products of a representative experiment. (D) Quantification of siRNA production from [32P]dsRNA relative to reaction in absence of rncs-1. The average of two sets of experiments with independent preparations of embryo extract is shown. Error bars, SEM.

 

Figure 4
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Fig. 4.. Dicer-regulated genes respond to rncs-1 levels. (A) Relative expression of Dicer-regulated genes in wild-type (gray) and rncs-1(tm1632) (white) adult hermaphrodites as quantified by qRT-PCR and normalized to gpd-3 (n = 6; error bars, SEM; *, P < 0.05, t test). (B) Relative expression of genes in A for the strains indicated. +, strains expressing the rncs-1 transgene; gfp, strain expressing Prncs-1::GFP. The mean value for three to six independent samples is plotted, normalized to let-413 (error bars, SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001, t test. Gray asterisks, comparison with rncs-1(tm1632); black asterisks, comparison with N2. (C) Fold change in mRNA level compared with wild-type is plotted for dcr-1(ok247) (circles), rncs-1(tm1632) (squares), and wild-type animals overexpressing rncs-1 (triangles). Data for dcr-1(ok247) are the average of qRT-PCR values for three or four independent samples. P < 0.001 for all genes (compared with wild type) except T07C5.1 (P < 0.05) and F35D11.3 (P < 0.01).

 

Figure 5
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Fig. 5.. rncs-1 alters small RNA production. (A) Northern blot of 3 µg of size-selected RNA from the indicated strains, probed for sequences complementary to F53A9.2 and C30F12.6. U6 RNA, loading control; black arrowhead, small RNA; white arrowhead, possible small RNA precursor. Putative precursor band in dcr-1 sample comigrates with that in (+) samples (data not shown). (B) As in A with strain designation as in Fig. 4. (C) Multiple analyses as in A and B were quantified to show small RNA levels in various strains relative to wild type. Results were normalized to U6 (F53A9.2: n = 2 for dcr-1, n = 4 for other strains; C30F12.6: n = 2 for all strains). Error bars, SEM. *, P < 0.05; ***, P < 0.01, t test.

 


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