Figure Legends for Supplementary Information
Supplementary Figure 1. Determination of the cleavage sites. a, Directional cloning of
pre-miR-30a. P32-radiolabeled pre-miR-30a was produced by in vitro transcription.
RNA in a size range of ~61-67-nt was purified and ligated to the 3’ and 5’ adapters.
Reverse transcription was performed using purified ligation product and reverse
transcription primer. To determine the 5’ end of pre-miR-30a, forward A that anneals to
the 5’ adapter sequence and reverse A that is complimentary to the 3’ region of premiR-30a were used for PCR amplification. After mapping the 5’ end, the 3’ end of premiR30a was determined by PCR using forward B and reverse transcription primer
(reverse B). Forward B primer contains 5’ adapter sequences as well as four nucleotides
(TGTA) from the 5’ end of pre-miR30a. b, The structure of the precursor for miR-30a.
Arrows indicate the cleavage sites.
Interestingly, the stem-loop contains a 2-nt overhang at its 3’ end, which is a
characteristic of the products of RNase III-mediated cleavage reaction. This 2-nt
3’overhang rule, however, may not apply to all pre-miRNAs. Pre-lin-4 from C. elegans,
which is the only pre-miRNA with known 5’ and 3’ ends, contains one noncomplementary nucleotide at each end1, indicating that cleavage occurs in the internal
loop without leaving staggered ends. In fact, it is not unusual for RNase III to cleave
internal loops or bulges or not to generate 2-nt 3’ overhangs. For instance, RNase III
from E. coli cleaves internal bulges or loops of many variants of T7 phage RNA without
leaving 2-nt 3’ overhang2. Pac1p from S. pombe can tolerate internal bulges and can
make a single-stranded cleavage3. In addition, S. cerevisiae Rnt1p cleaves not only U5
snRNA in an internal loop4 but also pre-rRNA at one strand of the stem5. Alternatively,
it is possible that C. elegans might use a different mechanism or other RNases trimmed
the overhang in C. elegans.
References
1.
Lee, R. C., Feinbaum, R. L. & Ambros, V. The C. elegans heterochronic gene
lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75,
843-54. (1993).
2.
Dunn, J. J. & Studier, F. W. Complete nucleotide sequence of bacteriophage T7
DNA and the locations of T7 genetic elements. J Mol Biol 166, 477-535. (1983).
3.
Rotondo, G., Huang, J. Y. & Frendewey, D. Substrate structure requirements of
the Pac1 ribonuclease from Schizosaccharmyces pombe. RNA 3, 1182-93.
(1997).
4.
Chanfreau, G., Elela, S. A., Ares, M., Jr. & Guthrie, C. Alternative 3'-end
processing of U5 snRNA by RNase III. Genes Dev 11, 2741-51. (1997).
5.
Elela, S. A., Igel, H. & Ares, M., Jr. RNase III cleaves eukaryotic preribosomal
RNA at a U3 snoRNP-dependent site. Cell 85, 115-24. (1996).
Supplementary Figure 2. Deletional mutagenesis on the precursor of miR-30a. Mutant
precursors were used for in vitro processing. Size markers (Decade marker, Ambion)
were loaded on lane M.
Supplementary Figure 3. Subcellular localization of Drosha. a, In vitro processing.
Subcellular fractionation of HEK293T cells was done in RSB-100 buffer (10 mM TrisHCl, pH7.5, 100 mM NaCl, 2.5 mM MgCl2, 35 g/ml digitonin). The resulting nuclear
(N) or cytoplasmic (C) fraction was used for immunoprecipitation with either nonimmunized rabbit sera (NRS) or rabbit anti-Drosha antibody (Drosha). Rabbit antiDrosha antibody was prepared by immunization with GST-Drosha protein containing
N-terminal half of Drosha. In vitro processing of let-7a-1 was performed with the
indicated immunoprecipitates. Size markers (Decade marker, Ambion) were loaded on
lane M. b, Western blot analysis. Nucleoplasmic and cytoplasmic fractions were
probed with anti-Drosha antibody, anti-hnRNP C1/C2 antibody, and anti-eIF4E
antibody.
Supplementary Figure 4. A model for miRNA biogenesis. MiRNA genes are transcribed
by an unidentified polymerase to generate the primary transcripts, referred to as primiRNAs. The initiation step (STEP 1) by Drosha results in pre-miRNAs of ~70nt,
which is exported by an unidentified export receptor. Upon export, Dicer participates in
the second step (STEP 2) to produce mature miRNAs. The final product may function
in a variety of regulatory pathways, such as translational control of certain mRNAs. The
question mark indicates the unidentified export factor.