Revealing Differential RNA Editing Specificity of Human ADAR1 and ADAR2 in Schizosaccharomyces pombe.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2's preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

Improving Effects of Peripheral Nerve Decompression Microsurgery of Lower Limbs in Patients with Diabetic Peripheral Neuropathy.

BACKGROUND: Peripheral nerve decompression microsurgery can relieve nerve entrapment and improve the symptoms of DPN. However, postoperative tissue adhesion will produce new pressure on the nerves, affecting the surgical efficacy. In this study, a nerve conduit was used in the peripheral nerve decompression microsurgery to prevent postoperative adhesions, and the role of the nerve conduit in surgical nerve decompression was explored. METHODS: A total of 69 patients with DPN were recruited and randomly divided into three groups: the nerve conduit group, conventional surgery group, and control group. Two weeks before surgery and 6 months after surgery, patients in each group were clinically tested using the visual analog scale (VAS) score, neurophysiological test, Toronto clinical scoring system (TCSS) score, and two-point discrimination (2-PD) test. RESULTS: The patients' symptoms in the nerve conduit group were relieved to varying degrees, and the relief rate reached 90.9%; the treatment efficacy was higher than that in the other groups. The postoperative nerve conduction velocity (NCV) in the two surgical groups was significantly higher than that before the surgery, and the difference between the nerve conduit group and the conventional surgery group was statistically significant (p < 0.05). For the 2-PD test, there was a statistically significant difference between the two surgical groups (p < 0.05). The TCSS score in the two surgical groups was significantly higher than that in the control group (p < 0.01). There was a significant difference in the TCSS scores between the nerve conduit group and the conventional surgery group (p < 0.05). CONCLUSIONS: The nerve conduit could further improve the efficacy of peripheral nerve decompression microsurgery in the treatment of DPN.

DeepEdit: single-molecule detection and phasing of A-to-I RNA editing events using nanopore direct RNA sequencing.

Single-molecule detection and phasing of A-to-I RNA editing events remain an unresolved problem. Long-read and PCR-free nanopore native RNA sequencing offers a great opportunity for direct RNA editing detection. Here, we develop a neural network model, DeepEdit, that not only recognizes A-to-I editing events in single reads of Oxford Nanopore direct RNA sequencing, but also resolves the phasing of RNA editing events on transcripts. We illustrate the robustness of DeepEdit by applying it to Schizosaccharomyces pombe and Homo sapiens transcriptome data. We anticipate DeepEdit to be a powerful tool for the study of RNA editing from a new perspective.

DUSP8/TAK1 signaling mediates neuropathic pain through regulating neuroinflammation and neuron death in a spinal nerve ligation (SNL) rat model.

Nerve injury-induced neuropathic pain is a type of chronic pain associated with neuroinflammatory response and neuronal death; however the underlying molecular mechanisms are still unclear. Dual-specificity phosphatase 8 (DUSP8) can mediate numerous cellular events, but whether it's involved in neuropathic pain is unknown. In the study, we found that spinal nerve ligation (SNL) operation on rats significantly decreased DUSP8 expression levels in ipsilateral spinal cord (ISC) tissues. Consistently, lipopolysaccharide (LPS) exposure also reduced DUSP8 in murine microglial cells. Adeno-associated virus (AAV)-mediated DUSP8 over-expression was found to considerably ameliorate SNL-induced neuropathic pain in rats. Additionally, neuronal death in the ISC tissues was also attenuated by AAV-DUSP8 following SNL surgery. Moreover, SNL-triggered neuroinflammation and microglial activation were also mitigated upon DUSP8 over-expression by suppressing nuclear factor κB (NF-κB) signaling, which were validated in LPS-exposed microglial cells. Importantly, our in vitro experiments indicated that inflammatory response in microglial cells contributed to neuron death, and such effect could also be ameliorated by DUSP8 over-expression. Notably, we found that DUSP8 directly interacted with transforming growth factor ß activated kinase-1 (TAK1) in microglial cells. Both SNL and LPS led to the activation of TAK1/p38/JNK1/2 signaling, whereas being strongly abolished by DUSP8. Intriguingly, TAK1 blockage significantly diminished LPS-induced inflammation and neuron death, whereas being accelerated by DUSP8 knockdown, further indicating that DUSP8-ameliorated neuropathic pain was largely TAK1-dependent. Together, all our findings revealed that DUSP8/TAK1 signaling may be a potential target for neuropathic pain alleviation.

Role of Mitophagy in the Pathogenesis of Stroke: From Mechanism to Therapy.

Mitochondria can supply adenosine triphosphate (ATP) to the tissue, which can regulate metabolism during the pathologic process and is also involved in the pathophysiology of neuronal injury after stroke. Recent studies have suggested that selective autophagy could play important roles in the pathophysiological process of stroke, especially mitophagy. It is usually mediated by the PINK1/Parkin-independent pathway or PINK1/Parkin-dependent pathway. Moreover, mitophagy may be a potential target in the therapy of stroke because the control of mitophagy is neuroprotective in stroke in vitro and in vivo. In this review, we briefly summarize recent researches in mitophagy, introduce the role of mitophagy in the pathogenesis of stroke, then highlight the strategies targeting mitophagy in the treatment of stroke, and finally propose several issues in the treatment of stroke by targeting mitophagy.

Controlled release of ciliary neurotrophic factor from bioactive nerve grafts promotes nerve regeneration in rats with facial nerve injuries.

It is critical to repair severed facial nerves, as lack of treatment may cause long-term motor and sensory impairments. Ciliary neurotrophic factor (CNTF) plays an important role in terms of enhancing nerve axon regrowth and maturation during peripheral nerve regeneration after injury. However, simple application of CNTF to the transected nerve site does not afford functional recovery, because it is rapidly flushed away by bodily fluids. The aim of the present study was the construction of a new, bioactive composite nerve graft facilitating persistent CNTF delivery to aid the reconstruction of facial nerve defects. The in vitro study showed that the bioactive nerve graft generated sustainable CNTF release for more than 25 days. The bioactive nerve graft was then transplanted into the injury sites of rat facial nerves. At 6 and 12 weeks post-transplantation, functional and histological analyses showed that the bioactive nerve graft featuring immobilized CNTF significantly enhanced nerve regeneration in terms of both axonal outgrowth and Schwann cell proliferation in the rat facial nerve gap model, compared to a collagen tube with adsorbed CNTF that initially released high levels of CNTF. The bioactive nerve graft may serve as novel, controlled bioactive release therapy for facial nerve regeneration.

Creating RNA Specific C-to-U Editase from APOBEC3A by Separation of Its Activities on DNA and RNA Substrates.

APOBEC3A (A3A) is a cytidine deaminase involved in innate immune response and is able to catalyze deamination on both DNA and RNA substrates. It was used in creating the CRISPR-mediated base editor, but has since been held back due to its dual activities. On the other hand, it has been a challenge to separate A3A's dual activities in order to enable it for single-base RNA editors. Here we developed the reporter system for C-to-U RNA editing and employed rational design for mutagenesis to differentiate deaminase activities on RNA and DNA substrates to obtain an RNA-specific editase. Generation and examination of 23 previous A3A mutants showed their deamination activity on RNA was mostly abolished when their activity on DNA was impaired, with the exception of mutant N57Q that displayed an inverse change. We designed new mutations on Loops 1 and 7 based on A3A's crystal structure and found mutants H29R and Y132G had differential effects on catalytic activity on RNA and DNA substrates. In order to engineer an A3A with RNA-specific deaminase activity, we combined Y132G with mutations in Loop 1 or helix 6 by rational design. Two multipoint mutants, Y132G/K30R and Y132G/G188A/R189A/L190A, were successful in retaining high deaminase activity on RNA substrate while eliminating deaminase activity on DNA. We, for the first time, created novel human A3A variants with RNA-specific cytidine deaminase activity, providing insight into A3A's mechanism on substrate recognition and a new addition of a toolset to the creation of a RNA-specific C-to-U base editor.

Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing.

In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.