Wirth, A. N. | bioRxiv Science

bioRxiv2026

Eukaryotic tRNA ligases mediate RNA break repair

RNA is continuously exposed to damage during physiological metabolism and stress, yet cellular responses to RNA damage remain less understood than DNA repair pathways. RNA strand breaks are particularly deleterious because they generate chemically incompatible RNA ends. Eukaryotic tRNA ligases have been implicated in RNA processing and repair, but whether they function as general RNA repair enzymes remains unresolved. Here, we show that the evolutionarily divergent tRNA ligases, human RTCB and fungal Trl1, mediate RNA break repair (RBR) targeting ribosomes and other ribonucleoprotein (RNP) complexes. Using direct RNA nanopore sequencing, we map these repair events at nucleotide resolution, demonstrating that tRNA ligases repair breaks in ribosomal RNA and restore translational activity of repaired ribosomes. We further identify repair across additional structured cellular RNAs. We show that loss of RBR activity leads to RNA fragmentation in human cells and impairs cell viability upon oxidative stress. Together, these findings uncover a broader role for eukaryotic tRNA ligases in repairing RNA breaks and maintaining transcriptome integrity.

biochemistry↗

bioRxiv2022

Adenosine Reduces Sinoatrial Node Cell AP Firing Rate by uncoupling its Membrane and Calcium Clocks

AO_SCPLOWBSTRACTC_SCPLOWThe spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase A (PKA) signaling. The mean AP cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine (ado) binds to adenosine receptors that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via G{beta}{gamma} signaling that activates an membrane-clock outward current, IKACh. Ado also inhibits adenylyl cyclase activity via Gi signaling, impacting cAMP-mediated PKA-dependent protein phosphorylation and intracellular Ca2+ cycling. We hypothesize that in addition to IKAdo activation, ado signaling impacts Ca2+ via Gi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 {micro}M ado substantially increased both the mean APCL (on average by 43%, n=10) and AP beat-to-beat variability from 5.1{+/-}1.7% to 7.2{+/-}2.0% (n=10) measured via membrane potential and 5.0{+/-}2.2 to 10.6{+/-}5.9 (n=40) measured via Ca2+ (assessed as the coefficient of variability, CV=SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic spontaneous, local Ca2+ releases (LCRs) (Ca2+ clock effect): as LCR size distributions shifted from larger to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR Ca2+ signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of ado "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher ado concentrations (>100 {micro}M): small stochastic LCRs of the Ca2+ clock failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of ado to activate G{beta}{gamma} and IKACh, Ado and to activate Gi, suppressing adenylyl cyclase activity, both contribute to the ado-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.

physiology↗