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. 2003 Nov 15;31(22):6593-7.
doi: 10.1093/nar/gkg855.

Human let-7 stem-loop precursors harbor features of RNase III cleavage products

Eugenia Basyuk  1 , Florence Suavet, Alain Doglio, Rémy Bordonné, Edouard Bertrand
Affiliations

Human let-7 stem-loop precursors harbor features of RNase III cleavage products

Eugenia Basyuk et al. Nucleic Acids Res. .

Abstract

The bidentate RNase III Dicer cleaves microRNA precursors to generate the 21-23 nt long mature RNAs. These precursors are 60-80 nt long, they fold into a characteristic stem-loop structure and they are generated by an unknown mechanism. To gain insights into the biogenesis of microRNAs, we have characterized the precise 5' and 3' ends of the let-7 precursors in human cells. We show that they harbor a 5'-phosphate and a 3'-OH and that, remarkably, they contain a 1-4 nt 3' overhang. These features are characteristic of RNase III cleavage products. Since these precursors are present in both the nucleus and the cytoplasm of human cells, our results suggest that they are generated in the nucleus by the nuclear RNase III. Additionally, these precursors fit the minihelix export motif and are thus likely exported by this pathway.

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Figures

Figure 1
Figure 1
(A) Schematic of the amplification strategy used to characterize let-7 minihelix precursors. (B) Secondary structure of human let-7a-3. Arrows indicate the oligonucleotides used for the RT–PCR. (C) Amplification products were resolved on a 1% agarose gel (upper panel) or a 12% acrylamide gel (lower panel) and stained with ethidium bromide. The RNA was treated before ligation as follows. Lane NT, no pre-treatment; lane Kin, T4 polynucleotide kinase; lane TAP, tobacco acid pyrophosphatase; lane –RT, reverse transcriptase was omitted.
Figure 2
Figure 2
(A) Structure of several of the human let-7 precursors. Arrows indictate the extremities of the stem–loop precursors characterized in this study. Clones of type A arose from let-7b (14% of the clones), clones B from let-7a-2 (14% of the clones) and clones C (large arrows, 58% of the clones) and D (small arrows, 14% of the clones) from either let-7a-1 or let-7a-3. (B) Nucleo-cytoplasmic distribution of let-7 stem–loop precursors. C, cytoplasmic RNAs; N, nuclear RNAs. Similar amounts of RNA were loaded in each lane. The RNA species probed is indicated on the left. (C) Consensus for the minihelix export motif (12). The length of the stem should be longer than 12 nt. Also note that the motif can tolerate many mismatches and bulges; the first base at the 5′ end can also be left unpaired.
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