Duane R. Wesemann
The enzyme tRNA 3’ Processing Endoribonuclease (3’tRNase) is suspected to be an enzyme responsible for 3’ end processing of various pre-tRNAs. Our lab has demonstrated that RNAs containing a hairpin structure can be manipulated to appear as a “pre-tRNA” molecule and be recognized and cleaved by 3’tRNase. This is accomplished by the introduction of an RNA molecule that is complementary to an area of the target RNA that has a stable hairpin structure. The hairpin of a target RNA may mimic the T-stem loop of tRNA and the hybridizing RNA molecule may act as an acceptor-like stem, thereby fooling 3’tRNase to cleave the target RNA. Specific degradation of various pre-tRNAs as well as non-tRNA ribonucleic acids (targeted by small guide RNAs) have been shown to be cleaved by 3’tRNase in vitro. Potential therapeutic strategies using post-transcriptional regulation of genes to combat cancer and disease exist if similar 3’tRNase activity can be demonstrated in vivo.
To demonstrate tRNA 3’ processing activity in vivo, a pre-tRNA substrate was injected into surgically extracted Xenopus laevis stage XI oocytes. The oocytes were incubated at room temperature for a number of time points (see figures). The RNA was extracted and separated on a 10% polyacrylamide denaturing gel next to the same pre-tRNA that was processed in vitro by 3’tRNase purified from pig liver. Three substrates were used to analyze 3’ processing activity in the oocytes. The substrate R-L0 is a radiolabeled, in vitro synthesized tRNAArg precursor that has a 13 nucleotide (nt) 3’ trailer and no 5’ leader. The substrate R-L6N is a radiolabeled, in vitro synthesized tRNAArg precursor that contains a 6 nt 5’ leader as well as a 3’ trailer. The substrate named T3H is a 55 nt radiolabeled RNA that contains a hairpin structure analogous to a T-stem of a tRNA. The guide RNA is a non-radioactive RNA molecule made to hybridize to T3H to cause the T3H + Guide RNA complex, a suitable substrate for 3’ tRNase and possibly other 3’ tRNA processing factors.
An autoradiograph of the in vitro processed R-L0 demonstrates a 13 nt cleavage product representing the cleaved 3’ trailer of the pre-tRNA. A similar band appears arising from the in vivo reaction of R-L0 at 90 minutes, indicating that some type of specific tRNA processing may be taking place within the oocyte (see figure 1A). The substrate R-L6N also seemed to be processed in vivo, showing bands similar to the ones arising from in vitro analysis of R-L6N 3’ processing. Both the 3’ unprocessed, as well as the 3’ processed tRNA molecules seem to be stable within the oocyte for up to 26 hours (see figure 1B). No significant specific degradation was observed by the in vivo incubations of the T3H + Guide complex. However, faint bands are observed that correspond to the cleavage product produced in vitro by purified 3’ tRNase (see figure 1C). Future experiments should include longer incubation times of Target + Guide complexes to determine more definitively if 3’ processing-related degradation is occurring.
Figure 1. (A) R-L0 tRNA precursor is cleaved by purified 3’ tRNase in vitro (lane 1). The same substrate was incubated in Xenopus oocytes for 10, 25, 40, and 90 minutes in lanes 2, 3, 4 and 5, respectively. (B) RL6N tRNA substrate cleaved by purified 3’ tRNase in vitro (lane 1). The same substrate was incubated in Xenopus oocytes for 10min., 25 min., 40min., 90min., 5 hr., 18 hr. and 28 hr. in lanes 2, 3, 4, 5, 6, 7, and 8 respectively. The upper band represents the tRNA precursor, the lower band the 3’ processed tRNA. (C) T3H target RNA incubated in vitro with purified 3’ tRNase (lane 1), and in Xenopus oocytes for 10, 25, 40, and 90 minutes in lanes 2, 3, 4 and 5, respectively. T3H is incubated with a small, non-radioactive guide RNA in vitro with purified 3’ tRNase (lane 6), as well as in Xenopus oocytes for 10, 25, 40, and 90 minutes in lanes 7, 8, 9 and 10 respectively.
The study represented here is an attempt to extend the phenomenon of targeted RNA degradation observed in vitro to an in vivo model using a small ribonucleotide chain to target the degredation of selected RNA substrates by endogenous RNase-dependent degredation. We show here that this may occur at a modest level in Xenopus oocytes. More studies need to confirm these results and to test the feasibility of extending this system to a mammalian model.
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