Temperature-dependent RNA editing in octopus extensively recodes the neural proteome

Abstract

In poikilotherms, temperature changes challenge the integration of physiological function. Within the complex nervous systems of the behaviorally sophisticated coleoid cephalopods, these problems are substantial. RNA editing by adenosine deamination is a well-positioned mechanism for environmental acclimation. We report that the neural proteome of Octopus bimaculoides undergoes massive reconfigurations via RNA editing following a temperature challenge. Over 13,000 codons are affected, and many alter proteins that are vital for neural processes. For two highly temperature-sensitive examples, recoding tunes protein function. For synaptotagmin, a key component of Ca²⁺-dependent neurotransmitter release, crystal structures and supporting experiments show that editing alters Ca²⁺ binding. For kinesin-1, a motor protein driving axonal transport, editing regulates transport velocity down microtubules. Seasonal sampling of wild-caught specimens indicates that temperature-dependent editing occurs in the field as well. These data show that A-to-I editing tunes neurophysiological function in response to temperature in octopus and most likely other coleoids.

Keywords: ADAR; RNA editing; RNA modifications; acclimation; cephalopod; epitranscriptome; kinesin; neural plasticity; synaptotagmin; temperature.

Figure 1:. Octopuses exposed to cold temperatures exhibit stronger RNA editing activity.

Panel A: Octopus bimaculoides (n=6 per temperature) were kept at 13 or 22°C for 12–24 days before dissecting stellate ganglia to measure A-to-I editing levels. Panel B: A large proportion of the O. bimaculoides editome exhibits increased editing at colder temperature (blue), but only 789 sites show a significant increase in warm samples (red). Panel C: Cold-induced increases in editing levels were both more common and higher in magnitude than warm-induced increases. See also Figure S1. Octopus drawings are reproduced, with permission, from Roger Hall.

Figure 2:. Cold-induced RNA editing sites are enriched in subtle, common amino acid substitutions.

Panel A: the majority of recoding editing sites do not show a statistically-significant temperature-sensitivity (grey) but a large proportion (33%, blue) are cold-induced and a small proportion are warm-induced (1%, red). Panel B: The fraction of recoding sites where amino acid substitutions stayed within the same polarity category is higher in cold-induced sites than warm-induced sites or sites with insignificant temperature sensitivity. The Bonferroni adjusted p-values from pairwise χ² tests are shown above each comparison. Panel C: The fraction of recoding sites resulting in evolutionarily common amino acid substitutions (positive BLOSUM scores) is higher in cold-induced sites than sites with insignificant temperature sensitivity. The Bonferroni adjusted p-values from pairwise t-tests on the raw BLOSUM scores are shown above each comparison. See also Figure S2.

Figure 3:. RNA editing changes within hours and reaches a steady state within days of a change in temperature.

Panels A and B: Octopuses were sampled before and at various timepoints after a 10°C transition that occurred over 20 hours. Panel C: Editing levels at 18 selected sites (see File S1) rise during a 10°C fall in temperature and at every timepoint afterwards. Each color represents a different editing site. The black line represents the mean editing level across all sites. The crosses represent the mean editing level from the long-term experiments at the equivalent ending temperature. Successive time points showing a statistically significant difference (see Table S5) are marked by distinct letters. Panel D: Editing levels at 18 selected sites decline during a 10°C rise in temperature and at almost every timepoint afterward. Colors and symbols are the same as described in panel C. Octopus drawings are reproduced, with permission, from Roger Hall.

Figure 4:. An editing site (K282R) on the motor domain of kinesin-1 is highly temperature-sensitive and induces strong changes in motility.

Panel A: Human monomeric kinesin (KIF5B) bound to tubulin (RCSB: 2P4N). Side chains are revealed for editing site and 10 conserved neighboring residues. Panel B: The K282R editing site is highly temperature-sensitive. Point data are shown from amplicon sequencing of a 4-day time-lapse experiment. Dotted horizontal lines represent editing levels during long-term temperature exposures at 13°C (blue) and 22°C (red). Panels C-E: Motility properties of individual wild-type (WT) and edited (K282R) octopus kinesin-1 along taxol-stabilized microtubules was visualized using single-molecule TIRF microscopy. From kymographs, the C) velocity, (D) run length, and (E) proportion of motile and stationary kinesins were determined. Motility properties were compared between wildtype (WT) and edited (K282R) kinesins at 21 and 11°C (*, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001). Panel E: The proportion of motile and stationary kinesins observed along microtubules were compared between WT and K282R and between temperatures. Error bars represent standard error. See also Figure S4.

Figure 5:. A cold-induced editing site (I248V) on the C2A domain of synaptotagmin-1 changes protein conformation to alter Ca²⁺-binding affinity.

Panel A: Wild-type (WT) Octopus bimaculoides synaptotagmin-1 C2A domain (blue, RCSB: 8FAF) and the I248V edited version (tan, RCSB: 8FAM) superimposed together. Top image shows the entire C2A domain including the Ca²⁺-binding region. The inset below zooms on residue 248 and the surrounding loop between β-strands 6 and 7, showing the change in conformation caused by the edit. See also Table S6. Panel B: The I248V editing site is highly temperature-sensitive. Point data are shown from amplicon sequencing of a 4-day time-lapse experiment. Dotted horizontal lines represent editing level during long-term temperature exposures at 13°C (blue) and 22°C (red). Panel C: Ca²⁺-binding affinity of the first (KD1) and second (KD2) Ca²⁺ ions to bind the C2A domain were determined via ITC for both the WT and I248V domains. **, p<0.01. Data are presented as mean +/− SD.

Figure 6:. Temperature-sensitive editing sites discovered in the laboratory show comparable temperature-sensitivity in wild-caught animals undergoing seasonal temperature.

Panel A: Kinesin-1 K282R and synaptotagmin-1 I248V editing sites present in O. bimaculoides caught in the wild both exhibit higher editing in February (15°C, blue) than in September (21°C, red), corroborating laboratory experiments. Panel B: The same temperature-dependent pattern is conserved in the sister species Octopus bimaculatus caught in the wild. **, p<0.01; ***, p<0.001; and ****, p<0.0001. See also Figure S5.