The RtcB RNA ligase is an essential component of the metazoan unfolded protein response

Abstract

RNA ligation can regulate RNA function by altering RNA sequence, structure and coding potential. For example, the function of XBP1 in mediating the unfolded protein response requires RNA ligation, as does the maturation of some tRNAs. Here, we describe a novel in vivo model in Caenorhabditis elegans for the conserved RNA ligase RtcB and show that RtcB ligates the xbp-1 mRNA during the IRE-1 branch of the unfolded protein response. Without RtcB, protein stress results in the accumulation of unligated xbp-1 mRNA fragments, defects in the unfolded protein response, and decreased lifespan. RtcB also ligates endogenous pre-tRNA halves, and RtcB mutants have defects in growth and lifespan that can be bypassed by expression of pre-spliced tRNAs. In addition, animals that lack RtcB have defects that are independent of tRNA maturation and the unfolded protein response. Thus, RNA ligation by RtcB is required for the function of multiple endogenous target RNAs including both xbp-1 and tRNAs. RtcB is uniquely capable of performing these ligation functions, and RNA ligation by RtcB mediates multiple essential processes in vivo.

Keywords: Caenorhabditis elegans; HSPC117; RtcB; tRNA; xbp‐1.

Figure 1

RtcB is required for ligation of endogenous intron-containing pre-tRNAs Northern blots of RNA extracted from wild-type and RtcB mutant worms, probed for tRNA^Leu(CAA). A tRNA halves accumulate in RtcB mutants (red arrowheads). B Levels of spliced mature tRNA^Leu(CAA) are reduced in RtcB mutants (green arrowheads). C Untrimmed, spliced pre-tRNA^Leu(CAA) intermediates are observed in wild-type but not RtcB mutants (yellow arrowheads). D Unspliced pre-tRNA^Leu(CAA) accumulates in RtcB mutants (blue arrowheads). E Quantitative analysis of the RNA species probed in (B) and (D), showing the relative amount in RtcB versus wild-type controls.

Figure 2

RtcB is required for survival under conditions of ER stress A Brightfield (top panels) and fluorescence (bottom panels) images of animals expressing the Phsp-4::GFP reporter. Animals were exposed to tunicamycin before imaging. Control animals (RtcB/+) exhibit strong GFP expression in multiple tissues; RtcB mutants fail to express GFP (faint signal is gut autofluorescence); expression of RtcB in the intestine of RtcB mutants rescues GFP expression specifically in intestinal cells. Scale bars, 100 μm. B Survival curves for wild-type animals (black), RtcB mutants (green), and RtcB mutants expressing pre-spliced tRNAs (blue). Filled symbols indicate normal growth conditions, and unfilled symbols indicate growth on tunicamycin. y-axis indicates the percentage of worms that survived. x-axis indicates the number of days after L4 stage. C Age-matched wild-type, RtcB mutant, and RtcB mutant worms expressing pre-spliced tRNAs. Scale bars, 100 μm. D Average length of wild-type (black), RtcB mutants (green), genomic rescued RtcB mutants (pink), and RtcB mutants expressing pre-spliced tRNAs (blue) at 48, 72, and 96 h after eggs were laid. N = 20 per genotype.

Figure 3

RtcB ligates the xbp-1 mRNA A RT–PCR using primers that detect both xbp-1^U and xbp-1^S. In wild-type, xbp-1^S is observed after 5 h of tunicamycin treatment. In RtcB nulls, xbp-1^S is not detected. B Expression of RFP-tagged wild-type RtcB and RFP-tagged inactive RtcB (H428A) in the intestine of RtcB/+ controls or RtcB mutant animals. Both wild-type and inactive RtcB are robustly expressed. Dashed boxes are detailed in (D). C Same animals as (B), visualizing GFP expression from the Phsp-4::GFP reporter under normal conditions (top panels) and after induction of ER stress (bottom panels). Expression of inactive RtcB (H428A) fails to rescue GFP induction in RtcB null mutants. Dotted circles indicate GFP expression in coelomocytes from the injection marker. Scale bars, 20 μm. D RFP-tagged RtcB is diffusely localized in the cytoplasm and nucleus. Images correspond to dotted squares in panel (B). Scale bars, 10 μm.

Figure 4

RtcB is required for the unfolded protein response A RNAseq read coverage at the xbp-1 locus in poly-A-selected, tunicamycin-treated, RtcB null worms. B Fold change per gene for genes that exhibit a ≥ 1.75 fold change in control worms, comparing untreated to tunicamycin-treated animals. Genes are ranked by fold change in control, shown in blue; pink bars indicate the fold change in RtcB mutants. C Same analysis as (B), except genes are selected that exhibit a ≥ 1.75 fold change in RtcB mutants. Genes are ranked by fold change in RtcB mutants, shown in pink; blue bars indicate the fold change in control animals. Data information: Data and gene names are listed in Supplementary Tables S2 and S3