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Fig. 4 | BMC Molecular Biology

Fig. 4

From: Conserved and highly expressed tRNA derived fragments in zebrafish

Fig. 4

Biogenesis of tRFs. 20 µg of total RNA were incubated with 2 U of recombinant Dicer or 1 µM of Angiogenin and was fractionated on 10 % PAGE, transferred to Hybond-N membranes and subjected to northern blot analysis. a 5′tRF-GluCTC and 5′tRF-ProCGG are produced by Dicer in vitro and its abundance increased over time, suggesting that the corresponding mature tRNAs are efficient Dicer substrates. Band intensity values normalized for the total RNA used are depicted for the tRFs generated over time by Dicer. For each tRF, the control RNA sample (first blot lane) was incubated without the enzyme for 6 h at 37 °C. The 3′tRFAlaAGC probe hybridized with the mature tRNA only and did not detect any tRF after Dicer incubation. b Analysis of 5′tRF-GluCTC and 5′tRF-ProCGG and 3′tRFAlaAGC production by Angiogenin showed unspecific cleavage of the mature tRNA, suggesting that Angiogenin was not involved in the biogenesis of any of the tRFs tested. c RNA shapes prediction of alternative structures of the mature tRNAs that originated 5′tRF-GluCTC and 5′tRF-ProCGG. The probabilities of formation of pre-miRNA hairpin-like structures (represented by []) is shown. The higher (>90 %) alternative folding probabilities of the 5′tRF-ProCGG precursors [tRNA(Pro)] is consistent with the higher cleavage efficiency of this tRNA, as shown in the northern blots in a. The tRNA(Ala)AGC does not originate tRFs after Dicer cleavage and its transcript folds as a typical mature tRNA. Sequences of tRFs of designed northern blot probes are highlighted in grey. The results of RNA shapes for each tRNA are shown below the secondary structures. The free energy, the probability of folding and the folding structures are depicted

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