RNA ligation in neurons by RtcB inhibits axon regeneration

Abstract

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) ...

Intrinsic mechanisms of neuronal axon regeneration

“…The RNA processing enzymes RNA 3´-terminal phosphate cyclases (RTCA and RTCB) act to inhibit axon regeneration after nerve injury in C. elegans and D. melanogaster 88,89 . By contrast, protein archease, an RNA ligase cofactor that functions downstream of RTCA, serves as a pro-regeneration factor 89 .…”

Axon regeneration in C. elegans: Worming our way to mechanisms of axon regeneration

“…In total, the screen identified more than 50 conserved genes that function in GABA neuron axon regeneration. Of these 50 genes, detailed analysis has been performed on the DLK-1 MAP Kinase pathway and its parallel MLK-1 MAP Kinase pathway, along with syndecan and the tRNA splicing ligase RTCB-1 have been characterized as regulators of axon regeneration (Hammarlund et al, 2009; Nix et al, 2011; Li et al, 2012; Edwards and Hammarlund, 2014; Kosmaczewski et al, 2015). The screen also led to the finding that Notch signaling inhibits axon regeneration (El Bejjani and Hammarlund, 2012).…”

The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans

“…RNA processing by the RtcB RNA ligase has also been implicated in axon regeneration (Kosmaczewski et al 2015). Loss of function in rtcb-1 enhances motor neuron regeneration, independent of known catalytic roles of RtcB in ligation of tRNAs or splicing of the mRNA for the unfolded protein response sensor xbp-1.…”