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. 2020 Sep 9;107(5):854-863.e6.
doi: 10.1016/j.neuron.2020.06.015. Epub 2020 Jul 7.

A Functional Non-coding RNA Is Produced from xbp-1 mRNA

Xiao Liu  1 , Jean-Denis Beaudoin  1 , Carrie Ann Davison  1 , Sara G Kosmaczewski  1 , Benjamin I Meyer  1 , Antonio J Giraldez  2 , Marc Hammarlund  3
Affiliations

A Functional Non-coding RNA Is Produced from xbp-1 mRNA

Xiao Liu et al. Neuron. .

Abstract

The xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) essential for axon regeneration in Caenorhabditis elegans. We show that the xbp-1 ncRNA acts independently of the protein-coding function of the xbp-1 transcript as part of a dual output xbp-1 mRNA stress response axis. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding, but not the coding, function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses.

Keywords: C. elegans; RNA processing; XBP1; axon regeneration; ncRNA; neuronal injury response; non-coding RNA; xbp-1.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. A processing intermediate of the xbp-1 mRNA promotes axon regeneration.
(A) Diagram of the xbp-1 mRNA splicing pathway. (B) RNA-seq analysis identifies non-canonical RNA junctions that are enriched in RtcB mutant animals compared to non-mutant controls. The Venn diagram plots genes with such RtcB-dependent junctions identified under normal condition (blue) or with neuronal injury (orange) at the indicated fold change cutoff. See also Table S1 and Table S2. (C) Animals with uncleavable xbp-1 fail to mount the UPR either in control conditions or upon tunicamycin treatment (5 μg/mL, 24 h). Scale bar, 50 μm. (D) Scheme of axotomy in C. elegans GABA neurons. DNC, dorsal nerve cord. VNC, ventral nerve cord. (E) Animals deficient of the RNA ligase RtcB show significantly higher regeneration. n = 37 and 44 from left to right. (F) Axon regeneration is eliminated in animals with the xbp-1(uncleavable) allele (see also Figure S1 for details about this allele). n = 44 and 43 from left to right. In (E) and (F), black bar represents the median. ***P<0.001, ****P<0.0001, 2-tailed K-S test.
Figure 2.
Figure 2.. The xbp-1 3’ fragment is necessary and sufficient to promote axon regeneration.
(A) The xbp-1 3’ UTR is required to increase regeneration in the rtcb-1(gk451); xbp-1(zc12) double mutant background. n = 62, 69, 40, and 31 from left to right. (B) Northern blot showing xbp-1 3’ fragment accumulation in RtcB mutant animals. (C) The xbp-1 3’ fragment is sufficient to increase regeneration cell-autonomously. n = 70 and 79 from left to right. (D) Deletion of the genomic xbp-1 locus eliminates regeneration, which is rescued by GABA-specific expression of the xbp-1 3’ fragment. n = 52, 92 and 34 from left to right. (E) Representative micrographs of animals of the indicated genotype 24 h post axotomy. Arrows indicate cut axons. Scale bar, 50 μm. In (A), (C) and (D), black bar represents the median. N.S., not significant, **P<0.01, ***P<0.001, 2-tailed K-S test.
Figure 3.
Figure 3.. The xbp-1 3’ UTR promotes axon regeneration as a ncRNA.
(A) The xbp-1 3’ UTR increases regeneration even when fused to GFP coding sequence. See also Figure S2D for representative micrographs showing GFP expression. To visualize GFP fluorescence, these constructs were expressed in a wild-type N2 background, as opposed to the oxIs12 background (GABA-specific GFP marker) used in other axotomy experiments. Regeneration was scored 14 h (instead of 24 h) after axotomy. n = 42 and 50 from left to right. See also Figure S2C and S2D. (B) When exported from the nucleus into the cytoplasm with the help of CTE, the xbp-1 3’ UTR increases regeneration without any coding sequence. n = 35 and 39 from left to right. (C) Diagrams of xbp-1 3’ fragment deletion constructs. (D) Axotomy results of animals expressing constructs in (C). n = 148, 39, 36, 57, and 48 from left to right. (E) Predicted secondary structure of the xbp-1 3’ fragment based on SHAPE-MaP results. (F) The xbp-1 3’ fragment with the RNA stem S134–209 deleted no longer promotes axon regeneration. n = 47 and 34 from left to right. In (A), (B), (D) and (F), black bar represents the median. N.S., not significant, *P<0.05, **P<0.01, ***P<0.001, 2-tailed K-S test.
Figure 4.
Figure 4.. A single base pair within S134–209 is essential for xbp-1 ncRNA function.
(A) Overexpression of the full-length xbp-1 transcript does not increase regeneration. n = 50, 41, and 45 from left to right. (B) Base-by-base comparisons of SHAPE reactivity difference across the length of S134–209. Arrows point to C159. (C) Predicted secondary structures of partial S134–209 based on SHAPE-MaP results. Left, the active forms (xbp-1 3’ fragment, CTE-UTR, and GFP-UTR). Right, the inactive form (full-length xbp-1). Box insert, diagram of point mutations used in (D-F). (D) Base pairing at C159 is essential for the overexpressed CTE::xbp-1 3’ UTR to increase regeneration. n = 67, 48 and 55 from left to right. (E) C159G mutation at the endogenous xbp-1 locus decreases regeneration. n = 49 and 50 from left to right. (F) The endogenous xbp-1 ncRNA impacts animal life span. n = 91, 93 and 116 from top to bottom. ****P<0.0001, log rank Mantel-Cox test. In (A), (D) and (E), black bar represents the median. N.S., not significant, *P<0.05, **P<0.01, 2-tailed K-S test. See also Figure S3.
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