*3.2.1 Gene silencing*

Similar to miRNAs, tRFs have been associated with the epigenetic regulator RISC, however mechanistic details on the role of tsRNA in the RISC remain to be elucidated. A recent meta-analysis of short RNA libraries from HEK293 cells demonstrated that both tRF-3 and tRF-5 associate with Ago proteins; however, a preference for Ago1, 3 and 4 over Ago2 was identified [59]. Interestingly a subsequent study in *D. melanogaster* revealed an age-related shift in tRF-Ago binding demonstrating a preference for Ago2 binding over Ago1 with increasing age [60].

### *3.2.2 Regulation of protein translation*

The synthesis of protein is a central activity in all cells that consumes a high level of energy and is dynamic in response to metabolic conditions and external stimuli. The regulation of protein translation therefore is a vital process in the maintenance of cell viability and the stress response. Canonical cap-dependent translation begins with the formation of the eukaryotic initiation factor (eIF) 4F complex containing eIF4A, a DEAD-box helicase, eIF4E and eIF4G. The eIF4E subunit binds to the 5' m7GTP cap on target mRNA and the eIF4G subunit is a scaffold protein that mediates the recruitment of other proteins including eIF3 and poly(A) binding protein (PABP). eIF4F binding to the 5′ m7GTP cap and the 3′ poly(A) tail circularizes the target mRNA and allows the 48S pre-initiation complex, containing the 40S small ribosomal subunit, Met-tRNAi met and eIF2, to scan the 5′ untranslated region and find the AUG start codon [61].

The dynamic regulation of protein translation in response to cellular stress and metabolic conditions is vital to cell survival. Stress-induced Ang-generated 5′tiRNA have been shown to halt the initiation of protein translation and facilitate the packaging of stalled translational complexes into stress granules [54]. Stress granules are cytoplasmic RNA-protein complexes that rapidly assemble and disassemble in response to cellular stress. This sequestration allows for the utilization of energy stores elsewhere and the recommencement of protein translation under optimum conditions [62]. Specific 5′tiRNA that contain a terminal oligoguanine (TOG) motif form stable G-quadruplex (G4) structures that directly bind the HEAT domain of eIF4G displacing eIF4A and inhibiting scanning of the mRNA target. Furthermore, 5′tiRNA with a 5′ monophosphate modification have been shown to bind the RNA binding protein YB-1 via the cold shock domain to precipitate the formation of stress granules [63].

Current knowledge on the effect of tRFs on protein translation is less advanced. Research in prokaryotic cells has demonstrated that tRF-5c of Val-GAC can bind the small ribosomal subunit and interfere with peptidyl transferase activity thereby inhibiting protein translation [64, 65]. In eukaryotic cells the tRF-3b of Gly-GCC reduced the level of specific protein with no concomitant reduction on mRNA levels indicating regulation at the translational level [66].

#### **3.3 tsRNA functions**

#### *3.3.1 tsRNA and apoptosis*

Disruption to the tsRNA system has been associated with increased cell death. Hypo-methylation of tRNA that arises from the inhibition of NSun2 increases cleavage by Ang and the accumulation of 5′tiRNA. The subsequent sustained depression in protein translation results in neuronal shrinkage, impaired synapse formation, cell death and is associated with neurodevelopmental deficiencies [67]. Loss-of-function mutations in the RNA kinase CLP1 has been shown to increase the level of tyrosine pre-tRNA fragments resulting in exaggerated p53 activation and vulnerability to cell death in cells exposed to oxidative stress [68]. Conversely, Ang has been shown to reduce cell death in neurons exposed to hyperosmotic stress in a tiRNA-mediated fashion. Specific Ang-generated tiRNA interact with cytochrome c and form a ribonucleoprotein complex that limits the formation of apoptosomes and reduces caspase-3 activation [53].
