Biochemical Dissection of tRNA Fragment Biogenesis

Transfer RNAs (tRNAs) are indispensable for decoding genetic information from messenger RNA into protein. Specific environmental insults induce the fragmentation of cellular RNAs, including tRNAs, which normally act as adaptor molecules during protein synthesis. tRNA fragmentation results in so-called tRNA fragments, which appear to be functional small RNAs and impact cells through mostly unknown molecular mechanisms. Various tRNA fragments have been repeatedly detected during cellular responses to stress such as starvation, oxidation, hypoxia and hypothermia as well as heat shock or irradiation. Importantly, specific tRNA fragments have been implicated in biological processes including proliferation, differentiation and translation, control of transposon activity, intercellular communication and transfer of extra-chromosomal information between generations. It is generally assumed that tRNA fragments are produced by the activity of enzymes, which introduce breaks into tRNAs. A single break in a tRNA should result in two tRNA fragments, which are often called tRNA halves. Interestingly though, cells recovering from stress only maintain one tRNA half, which indicates the presence of cellular activities that degrade the other tRNA half. This project will identify the enzymatic activities, which act on tRNAs that were broken during the stress response of human cells. Specifically, the project aims at identifying enzymes, which degrade only one tRNA half in broken tRNAs while leaving the other intact. To do so, the project will employ biochemical fractionation of protein complexes induced by experimental stress conditions in human cell culture systems. Once identified, the molecular details defining these activities will be determined by measuring enzymatic reaction parameters on broken tRNAs using recombinant protein and RNA approaches. Given the widespread detection of tRNA fragments in many species, their functional association with specific cellular processes and potential to serve as biomarkers for particular human diseases, identifying the complete molecular machinery that produces tRNA fragments is likely to have a major impact on our understanding as to how these small RNAs exert biological impact. Furthermore, identifying the enzymes whose function impacts the production and thereby likely the function of tRNA fragments will provide molecular insights that could inform diagnostic and even therapeutic avenues connected to utilizing these small RNAs.

Transfer RNAs (tRNAs) are indispensable for reading mRNAs and their translation into protein sequences. tRNAs can also become substrates of dedicated enzymes, especially during the cellular stress response, resulting in the breaking of the tRNA backbone at specific sites. The products of such tRNA cleavage are known as tRNA-derived RNAs (tDRs). tDRs have been associated with specific molecular pathways and diseases, evoking great interest in their biological impact and their potential as biomarkers for tissue injury and cellular stress. However, a large knowledge gap in the current understanding of tDR biology remained for a long time. Since tRNAs are highly structured molecules, it was unclear how the hydrolysis of one phosphodiester bond, resulting in "nicked" tRNAs, would be sufficient for the production of individual and biologically active tDRs. Overlapping with the start of this project, we had previously identified specific human RNA helicases that can unwind "nicked" tRNAs. This finding formed the basis for the molecular dissection of tDR biogenesis, namely addressing the phenomenon of tDR asymmetry, the unequal production of 5' and 3' tDRs from the same parental tRNA. To do so, biochemical approaches were employed to systematically fractionate the cellular proteome of human cells, and testing of these fractions for 3' tDR-degrading activities using in vitro assays. In parallel, RNases that associated with the anticodon nuclease Angiogenin, the main enzyme responsible for "nicking" tRNAs during the stress response, were identified by immunoprecipitation followed by mass spectrometry. These approaches led to the identification of several RNases that can process stress-induced 3 tDRs. Specifically, RNA exosome components were physically associated with "nicked" tRNAs and exerted ribonuclease activity on 3 tDRs in vitro. The results of these experiments are currently being validated and extended by interference with the in vivo function of the identified RNases. Taken together, the findings from this project indicated that upon initial stress-induced tRNA fragmentation in human cells, "nicked" tRNAs are not only separated into 5' and 3' tDRs by dedicated RNA helicases, but also that 5' tDRs are selectively maintained at the expense of the degradation of 3' tDRs, which suggests that 5' tDRs are singled out for potential biological activity.