After axonal injury, chromatolysis (fragmentation of Nissl substance) occurs in both intrinsic neurons (whose processes are within the CNS) and extrinsic neurons (whose axons extend outside the CNS). Electron microscopy shows that chromatolysis involves fission of the rough endoplasmic reticulum. In intrinsic neurons (which do not regenerate axons) or in extrinsic neurons denied axon regeneration, chromatolysis is often accompanied by degranulation (loss of ribosomes from rough endoplasmic reticulum), disaggregation of polyribosomes and degradation of monoribosomes into dust-like particles. Ribosomes and rough endoplasmic reticulum may also be degraded in autophagic vacuoles by Ribophagy and Reticulophagy, respectively. In other words, chromatolysis is disruption of parts of the protein synthesis infrastructure. Whereas some neurons may show transient or no chromatolysis, severely injured neurons can remain chromatolytic and never again synthesise normal levels of protein; some may atrophy or die. What molecule(s) cause fragmentation or degranulation of rough endoplasmic reticulum, disaggregation of polyribosomes and degradation of monoribosomes? Ribonucleases can modify (and perhaps fragment) rough endoplasmic reticulum; various endoribonucleases can degrade mRNA causing polyribosomes to unchain and disperse; they can disassemble monoribosomes; Ribonuclease 5 can control rRNA synthesis and degrade tRNA; Ribonuclease T2 can degrade ribosomes, rough endoplasmic reticulum and RNA within autophagic vacuoles; and Ribonuclease IRE1α acts as a stress sensor within the endoplasmic reticulum. Regeneration might be improved after axonal injury by protecting the protein synthesis machinery from catabolism; targeting ribonucleases could be a profitable strategy.

Protein oligomerzation is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads abnormal protein oligomerization resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson’s disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants: H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near physiological conditions. All three variants are found to adopt well folded and highly rigid structures in solution, although the elements of secondary structure are somewhat shorter than those observed in cystallography studies. R121C alters the environment of nearby residues only. By constrast, the mutation H13A affects local residues as well as nearby active site residues residues K41 and H119. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of most duplex structure and little or no G-quadruplex formation.

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification, resulting in angiogenin (ANG) dependent tsRNA formation. More specifically, m5U54 hypomodification is followed by ANG overexpression and tRNA cleavage near the anticodon, resulting in accumulation of 5’tRNA-derived stress-induced RNAs (5’tiRNAs), in particular 5’tiRNA-GlyGCC and 5’tiRNA-GluCTC. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tsRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tsRNA formation in mammalian cells. These results establish a link between tRNA demethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.