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Engineering of a conditional allele reveals multiple roles of XRN2 in Caenorhabditis elegans development and substrate specificity in microRNA turnover - PubMed

Engineering of a conditional allele reveals multiple roles of XRN2 in Caenorhabditis elegans development and substrate specificity in microRNA turnover

Takashi S Miki et al. Nucleic Acids Res. 2014 Apr.

Abstract

Although XRN2 proteins are highly conserved eukaryotic 5'→3' exonucleases, little is known about their function in animals. Here, we characterize Caenorhabditis elegans XRN2, which we find to be a broadly and constitutively expressed nuclear protein. An xrn-2 null mutation or loss of XRN2 catalytic activity causes a molting defect and early larval arrest. However, by generating a conditionally mutant xrn-2ts strain de novo through an approach that may be also applicable to other genes of interest, we reveal further functions in fertility, during embryogenesis and during additional larval stages. Consistent with the known role of XRN2 in controlling microRNA (miRNA) levels, we can demonstrate that loss of XRN2 activity stabilizes some rapidly decaying miRNAs. Surprisingly, however, other miRNAs continue to decay rapidly in xrn-2ts animals. Thus, XRN2 has unanticipated miRNA specificity in vivo, and its diverse developmental functions may relate to distinct substrates. Finally, our global analysis of miRNA stability during larval stage 1 reveals that miRNA passenger strands (miR*s) are substantially less stable than guide strands (miRs), supporting the notion that the former are mostly byproducts of biogenesis rather than a less abundant functional species.

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Figures

Figure 1.
Figure 1.

xrn-2(tm3473) is a bona fide null allele that causes molting defects and developmental arrest. (A) Schematic representation of wild-type and mutant XRN2. Conserved regions are shown in light grey. Dark grey indicates sequence unique to the xrn-2(tm3473) mutant due to a frame shift. Point mutations investigated in this study are indicated. (B) Western blotting confirms absence of endogenous XRN2 in the xrn-2(tm3473) background (lane 3). xrn-2(+) denotes the N2 wild-type strain. Note the presence of an XRN2/GFP-encoding transgene in the strains shown in lane 2 and 3, used to restore development of the xrn-2(tm3473) mutant strain. (C) DIC micrographs of worms grown at 25°C; gonads are outlined to facilitate staging. (i, ii) After 18 h, both xrn-2/+ (tm3473 heterozygous) and xrn-2/xrn-2 (tm3473 homozygous) worms are at the L2 stage. (iii, iv) After 29 h, xrn-2/xrn-2 worms remain arrested at the L2 stage (iv), whereas the heterozygous siblings have reached the L4 stage (iii). (v, vi) Larval arrest is accompanied by molting defects. xrn-2/xrn-2 worms are unable to shed the pharyngeal cuticle (v, arrow head), which leads to superposition of the old and newly synthesized cuticle (vi, arrow heads). Scale bar, 20 µm.

Figure 2.
Figure 2.

XRN2 catalytic activity is required for molting and growth beyond the L2 stage. (A) Wild-type worms develop into gravid adults, whereas (B) xrn-2(tm3473) homozygous worms arrest development. (C) Transgenic extrachromosomal xrn-2 expressed under the control of the xrn-2 1413-bp promoter and xrn-2 3′-UTR rescues xrn-2(tm3473) mutant animals, whereas (D) a catalytically inactive version of xrn-2 with two point mutations (D234A and D236A) does not. Both transgenes contain a C-terminal GFP tag, permitting their detection with an anti-GFP antibody. (E) Western blotting reveals equivalent accumulation of wild-type (lane 1) and mutant (lane 2) protein in vivo. Scale bar, 50 µm. xrn-2(+) denotes the N2 wild-type strain.

Figure 3.
Figure 3.

XRN2 is ubiquitously and constitutively expressed. (A) Schematic depiction of the xrn-2 genomic locus and promoters used. The arrows indicate the direction of transcription. (B) Micrographs showing GFP signal of single-copy-integrated, codon-optimized and gfp-tagged xrn-2 expressed under the control of the 1413-bp long promoter region. The GFP signal is ubiquitously detected. Examples of hypodermal and intestinal cells are marked with arrowheads. Insets: DIC images of the same worms. (C) Western blot showing a time-course for endogenous XRN2. ‘e’, ‘m’ and ‘l’ stands for early, mid and late, respectively; ‘YA’ and ‘GA’ for young and gravid adult, respectively. An asterisk indicates an apparent proteolytic fragment of XRN2, which did not occur consistently in other western blots. (D) Single-copy-integrated, codon-optimized and gfp-tagged xrn-2 expressed under the control of the 1413-bp long xrn-2 promoter region rescues the phenotypes of xrn-2(tm3473), but the 132-bp long xrn-2 promoter region does not. Scale bar, 20 µm (B) and 50 µm (D).

Figure 4.
Figure 4.

Characterization of an improved xrn-2ts strain reveals reduced XRN2 levels at restrictive temperature. (A) Schematic representation of xrn-2ts phenotypes at different temperature. xrn-2ts embryos or worms were cultured under the indicated conditions. Phenotypes observed are described on the right. For less-penetrant phenotypes, a number indicating worms affected/worms scored is shown in brackets. (B) The wild-type (‘wt’; N2) and xrn-2ts worms were cultured from L1-stage at 20 or 26°C as indicated for 72 h (wt) and 93 h (xrn-2ts), respectively. The worms were observed by stereo microscopy at the same magnification. (C, D) The wt, xrn-2(+) and xrn-2ts worms were cultured from mid L3- to late L4-stage at 15°C or 26°C and harvested. (C) XRN2, XRN2/GFP and actin protein levels were examined by western blotting. XRN2 and XRN2/GFP levels were normalized to actin levels and shown with values of wt at 15°C defined as 100. (D) The mRNA levels of the xrn-2::gfp transgenes in xrn-2(+) and xrn-2ts worms were quantified by RT-qPCR, normalized to actin mRNA levels and shown with values of xrn-2(+) at 15°C as 1 (n = 2, means + SEM). ‘xrn-2(+)’ denotes xrn-2(tm3473) homozygous animals expressing a wild-type xrn-2 transgene.

Figure 5.
Figure 5.

Specific miRNAs are stabilized on XRN2 depletion. (A) Experimental design for miRNA decay analysis. RNA was extracted for (B) deep sequencing and (C) RT-qPCR analyses. (B) Relative decay rates of miRNAs are plotted against normalized reads for miRNAs with sufficient expression (‘Materials and Methods’ section). Black stars, miRs; red stars, miR*s. Fifteen miRs that showed high-read numbers and fast relative decay as indicated by the blue cut-off lines. (Because the plot shows fold changes per hour, not decay constants, unstable miRNAs are those below the cut-off line.) These and two miR*s, indicated in black, were further examined by RT-qPCR. Other miRs discussed in the main text are shown in grey. (C) The miRNA levels at each time point in wt and xrn-2ts worms were quantified by RT-qPCR, and their half-lives were calculated as described in ‘Materials and Methods’ section. Relevant P-values are shown. ns, not significant.

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