C. elegans and H. sapiens mRNAs with edited 3' UTRs are present on polysomes - PubMed (original) (raw)

C. elegans and H. sapiens mRNAs with edited 3' UTRs are present on polysomes

Heather A Hundley et al. RNA. 2008 Oct.

Abstract

Adenosine deaminases that act on RNA (ADARs) are editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). ADARs sometimes target codons so that a single mRNA yields multiple protein isoforms. However, ADARs most often target noncoding regions of mRNAs, such as untranslated regions (UTRs). To understand the function of extensive double-stranded 3' UTR structures, and the inosines within them, we monitored the fate of reporter and endogenous mRNAs that include structured 3' UTRs in wild-type Caenorhabditis elegans and in strains with mutations in the ADAR genes. In general, we saw little effect of editing on stability or translatability of mRNA, although in one case an ADR-1 dependent effect was observed. Importantly, whereas previous studies indicate that inosine-containing RNAs are retained in the nucleus, we show that both C. elegans and Homo sapiens mRNAs with edited, structured 3' UTRs are present on translating ribosomes.

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Figures

FIGURE 1.

FIGURE 1.

mRNA levels of C. elegans ADARs. Points represent average mRNA level in RNA isolated from two independent developmental time-course experiments using wild-type worms. Relative expression of adr-1 (circle, dashed line) and adr-2 (square, solid line) was quantified by qRT-PCR (error bars=SEM) with normalization to the average of three genes, gpd-3, ama-1, and fasn-1, that were previously determined to be expressed at relatively constant levels throughout development (Jiang et al. 2001). _Y_-axes are 10-fold different and indicate the relative level of adr-1 (left) and adr-2 (right) mRNA. _X_-axis denotes worm stages as embryo (E), larval (L1–L4), and young adult (YA).

FIGURE 2.

FIGURE 2.

In vivo reporter assay for mRNAs with edited 3′ UTRs. (A) Northern blots were performed on total RNA from wild-type strains expressing both rab3∷gfp∷unc-54 (3′ UTR) and the following: (Lane 1) rab3∷rfp∷C35E7.6 (3′ UTR), (lane 2) rab3∷rfp∷unc-64 (3′ UTR), (lane 3) rab3∷rfp∷elo-3 (3′ UTR), (lane 4) rab3∷rfp∷pop-1 (3′ UTR), (lane 5) rab3∷rfp∷lam-2 (3′ UTR). Blots were hybridized with a probe for the rfp coding sequence. (B) Nomarski, GFP, and RFP images (left to right) of the wild-type strain expressing the transgenic rab3∷gfp∷unc-54 (3′ UTR) and rab3∷rfp∷C35E7.6 (3′ UTR) reporters. (C_–_F) For various strains, the height of the bar represents the fluorescence intensity of reporter proteins synthesized from mRNA with structured 3′ UTRs relative to that derived from mRNA with the control 3′ UTR, normalized to a wild-type (WT) value of 1. Error bars show SEM for multiple trials, where each trial (n) included 13 young adult worms. Analyses were performed on worms expressing the following transgenes: (C) rab3∷rfp∷elo-3 (3′ UTR) and rab3∷gfp∷unc-54 (3′ UTR), n = 3; (D) rab3∷rfp∷unc-64 (3′ UTR) and rab3∷gfp∷unc-54 (3′ UTR), n = 2; (E) rab3∷gfp∷lam-2 (3′ UTR) and rab3∷rfp∷unc-54 (3′ UTR), n = 1; (F) rab3∷rfp∷C35E7.6 (3′ UTR) and rab3∷gfp∷unc-54 (3′ UTR), n = 3. (*) Significant differences of P = 0.001. Both sets of adr alleles were used for analyzing reporters of elo-3, unc-64, and C35E7.6, and were found to give similar results; thus, only a single set of alleles was used in the lam-2 analysis. In C, D, and F, data shown for adr-1 were performed with the tm668 allele, and for _adr_-2 with the ok735 allele; in E, adr-1 and adr-2 indicate the gv6 and gv42 alleles, respectively.

FIGURE 3.

FIGURE 3.

Polysome association of C. elegans transgenic mRNAs containing structured 3′ UTRs. Cytoplasmic extracts from young adult worms were isolated in the presence of the translation inhibiting drug, cycloheximide, (A, C_–_G) or EDTA (B) and separated by sucrose density sedimentation. An absorbance trace (254 nm) across the gradient appears above Northern blots of total RNA isolated from each fraction. The particular structured 3′ UTR being monitored is indicated by the transgene name in the upper right corner of each UV trace. In A and B, mRNA for the structured 3′ UTR, rab3∷rfp∷C35E7.6 (3′ UTR), and control 3′ UTR, rab3∷gfp∷unc-54 (3′ UTR), were both monitored; the upper Northern blot was hybridized with a probe for the rfp coding sequence (structured 3′ UTR), stripped, and re-hybridized with a probe for the gfp coding sequence (lower blot; control 3′ UTR). Blots for the transgenic mRNAs with structured 3′ UTRs monitored in C_–_G were hybridized with a probe for the gfp coding sequence (C) or the rfp coding sequence (D_–_G).

FIGURE 4.

FIGURE 4.

Levels of C. elegans mRNAs containing structured 3′ UTRs in animals deficient for ADARs. (A) Relative mRNA expression in embryos was quantified by qRT-PCR in the following strains: wild-type (black), adr-1(tm668) (light gray), adr-2(ok735) (white), and adr-1(tm668);adr-2(ok735) (dark gray). Graph illustrates the average fold-change of each mRNA relative to gpd-3 mRNA and normalized to the wild-type level (n = 4, error bars=SEM). (B) Relative mRNA expression as in A for the young adult stage.

FIGURE 5.

FIGURE 5.

Polysome association of endogenous C. elegans mRNAs containing edited 3′ UTRs. Cytoplasmic extracts from wild-type young adult worms were isolated in the presence of cycloheximide (A) or EDTA (B) and separated by sucrose density sedimentation. The absorbance trace at 254 nm was monitored for each gradient (top panel) and mRNA levels quantified for each fraction (lower graphs). mRNA levels were determined using qRT-PCR for C35E7.6, unc-64, and gpd-3 and plotted as the fraction of mRNA present. (C) C. elegans genomic DNA, cDNA from total RNA, and cDNA from the polysome portion of the gradient were amplified and sequenced to detect editing events in the 3′ UTRs of C35E7.6 and unc-64. Data for fraction 9 are shown, but editing was detected in fractions 7–11 (data not shown). (D) The percents of total unc-64a (dark gray) and unc-64b (light gray) mRNA, quantified by qRT-PCR for two independent polysome profiles, were plotted against the number of ribosomes in each fraction (error bars=SEM).

FIGURE 6.

FIGURE 6.

Mammalian mRNAs with edited 3′ UTRs are present on polysomes. Cytoplasmic extracts from HeLa cells were isolated in the presence of cycloheximide (A) or EDTA (B) and separated by sucrose density sedimentation. mRNA levels were determined by qRT-PCR for NDUFC2 and PSMB2 and GAPDH and plotted as described in Fig. 5. (C) HeLa genomic DNA, cDNA from total RNA, and cDNA from the polysome portion of the gradient were amplified and sequenced to detect the editing events present in the 3′ UTRs of NDUFC2 and PSMB2. Data for fraction 9 are shown, but editing was detected in fractions 7–11 (data not shown).

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