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.

Neurodegeneration has been correlated with mitochondrial DNA (mtDNA) damage and exposure to environmental toxins, but causation is unclear. We investigated the ability of several known environmental genotoxins and neurotoxins to cause mtDNA damage, mtDNA depletion, and neurodegeneration in Caenorhabditis elegans. We found that paraquat, cadmium chloride and aflatoxin B1 caused more mitochondrial than nuclear DNA damage, and paraquat and aflatoxin B1 also caused dopaminergic neurodegeneration. 6-hydroxydopamine (6-OHDA) caused similar levels of mitochondrial and nuclear DNA damage. To further test whether the neurodegeneration could be attributed to the observed mtDNA damage, C. elegans were exposed to repeated low-dose ultraviolet C radiation (UVC) that resulted in persistent mtDNA damage; this exposure also resulted in dopaminergic neurodegeneration. Damage to GABAergic neurons and pharyngeal muscle cells was not detected. We also found that fasting at the first larval stage was protective in dopaminergic neurons against 6-OHDA-induced neurodegeneration. Finally, we found that dopaminergic neurons in C. elegans are capable of regeneration after laser surgery. Our findings are consistent with a causal role for mitochondrial DNA damage in neurodegeneration, but also support non mtDNA-mediated mechanisms.

Activity of the RNA ligase RtcB has only two known functions: tRNA ligation after intron removal and XBP1 mRNA ligation during activation of the unfolded protein response. Here, we show that RtcB acts in neurons to inhibit axon regeneration after nerve injury. This function of RtcB is independent of its basal activities in tRNA ligation and the unfolded protein response. Furthermore, inhibition of axon regeneration is independent of the RtcB cofactor archease. Finally, RtcB is enriched at axon termini after nerve injury. Our data indicate that neurons have co-opted an ancient RNA modification mechanism to regulate specific and dynamic functions and identify neuronal RtcB activity as a critical regulator of neuronal growth potential.axon regeneration | RNA ligation | RtcB T he RNA ligase RtcB is the only known RNA ligase in metazoans. RNA ligation by RtcB is required for the maturation of intron-containing tRNAs (1-3), and also, it is required to process the transcription factor xbp-1 mRNA and activate the unfolded protein response (UPR) (4-6). Other than these two basic cellular processes, which are likely common to all metazoan cells, no functions for RNA ligation or RtcB are known. The nervous system is a site of expanded RNA processing after transcription. For example, neurons regulate alternative premRNA splicing in response to activity (7-10) and are highly enriched for mRNA editing (11-13). Here, we define a neuron-specific function for RtcB activity in regulating axon regeneration and show that this neuronal function is independent of RtcB's activities in tRNA and xbp-1 ligation.RtcB activity in neurons inhibits axon regeneration. We assayed axon regeneration in the GABA motor neurons of Caenorhabditis elegans using single-neuron laser axotomy (14). Mutants in the single C. elegans ortholog of RtcB, rtcb-1(gk451) (5), exhibited increased regeneration to the dorsal nerve cord (DNC) at 24 h after injury, consistent with previous data (Fig. 1 A and B) (15). Increased DNC regeneration was reduced to WT levels by introduction of a single-copy WT RtcB transgene (Fig. 1A). The increase in DNC regeneration is not caused by the trivial explanation that RtcB animals are narrower than the WT. At 6 h after injury, a time point at which neurons in WT animals are just initiating regeneration (15-17), a substantial fraction of axons in RtcB mutants had already regenerated to the DNC (Fig. 1C). Furthermore, WT animals did not regenerate as well as RtcB mutants, even when given additional time to regenerate (Fig. 1C). Finally, rescuing the overall growth defects of RtcB mutants did not alter the effect of loss of RtcB on axon regeneration (Fig. 2). Thus, loss of RtcB results in faster and more successful axon regeneration. Increased regeneration depends on loss of RtcB in neurons, because expressing WT RtcB under a GABA-specific promoter restored DNC regeneration to WT levels (Fig. 1A). DNC regeneration levels were not restored when the rescue construct contained a point mutation that eliminates ligase activity (H428A) ...

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) in Caenorhabditis elegans neurons, providing the first example of ncRNA derived from mRNA cleavage. We show that the xbp-1 ncRNA is crucial for axon regeneration in vivo, and that it acts independently of the proteincoding function of the xbp-1 transcript. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; and this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding but not 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.

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 10 cleavage is a biologically active non-coding RNA (ncRNA) in Caenorhabditis elegans neurons, providing the first example of ncRNA derived from mRNA cleavage. We show that the xbp-1 12 ncRNA is crucial for axon regeneration in vivo, and that it acts independently of the proteincoding function of the xbp-1 transcript. Structural analysis indicates that the function of the xbp-141 ncRNA depends on a single RNA stem; and this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding but not coding function of the 16 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 18 response to injury and other stresses.Graphic abstract: 20 22