The loop region of Let-7 interacts with the Drosha Inhibitor. (A) The sequence alignment of Let-7 family members is shown. (Blue text) Mature miRNA sequence; (green text) the complementary stem strand (not exactly the star strand); (gray boxes) regions of homology; (arrows) nucleotides that were mutagenized. Sequence changes are indicated below the alignment. (B) Wild-type (WT) or mutant pri-Let-7g substrates were combined with the pri-miR-17 substrate and incubated in a P19 or HeLa nuclear extract. Drosha products were resolved and are indicated. (C) Pri-miRNA substrates for Let-7g and miR-17 were combined and incubated in P19 nuclear extracts. Competitor RNA transcripts corresponding to the loop plus 12 nt of each stem, or competitor 2′-O-methyl oligonucleotides, were included at 10, 50, and 250 nM final concentration. In one case, the oligonucleotide had a 3′ Biotin moiety. The left lane had no competitor. Drosha products were resolved on a denaturing polyacrylamide gel. (Arrows) Precursor products.

Lin-28 expression blocks production of Let-7. (A) NIH-3T3 cells were transduced with MSCV retroviral constructs that drive expression of Lin-28, Lin-28B, or control, as indicated. Steady-state miRNA expression levels were quantitated using a custom microarray platform 10 d post-infection. Normalized measurements were hierarchically clustered and are plotted as a heat map. (Yellow) High expression, (blue) low expression, relative to the mean. (Red font) Let-7 family members. (B) Steady-state miRNA expression levels from NIH-3T3 cells expressing Lin-28, Lin-28B, or control were quantitated by real-time RT-PCR. U6 snRNA was used as the reference. Expression of pri-Let-7g was also quantitated by real-time RT-PCR. β2-microglobulin was used as the reference. (C) Let-7g precursor and mature species in NIH-3T3 cells expressing Lin-28, Lin-28B, or control were analyzed by Northern blotting. (D) P19 cells were transfected with siRNAs targeting Lin-28. Two effective siRNAs were used alone or in combination. Five days post-transfection, mature miRNA levels were measured by real-time RT-PCR. U6 was used as a reference. Expression of pri-Let-7g was also quantitated by real-time RT-PCR. β2-microglobulin was used as the reference.

Extensive sequence conservation of pri-Let-7. The genomic locus of the mouse pre-Let-7g and pre-Let-7i stem–loop sequence are shown, with 600 nt flanking on each side included. The conservation plot of 30 vertebrate species is shown. Data were obtained from the UCSC genome browser, July 2007 release.

Embryonic cells contain a Drosha Inhibitor that specifically regulates Let-7. (Left) Radiolabeled pri-miRNA substrates corresponding to Let-7g and miR-17 were incubated in P19 or HeLa nuclear extracts, as indicated. (Right) Drosha protein, or mock, was immunoprecipitated from P19 nuclear extracts with a polyclonal antibody. Immobilized protein was incubated with Let-7g and miR-17 pri-miRNA substrates, as indicated. Drosha endonuclease products were resolved on a denaturing polyacrylamide gel. (Arrows) Production of Let-7g and miR-17 precursors. A labeled RNA oligonucleotide ladder is shown for size reference.

The RNA binding protein Lin-28 specifically binds to the Let-7 loop region. (A) Oligonucleotide capture probes corresponding to the Let-7d loop or a random (control) sequence, fully 2′-O-methyl modified, 3′ Biotin linked, were bound to streptavidin agarose. Proteins were captured from P19 nuclear extracts, were resolved on a 4%–20% polyacrylamide gel, and were stained with coomassie blue. Proteins were isolated and identified by MALDI-TOF fingerprinting. (B) Radiolabeled RNA probes corresponding to the Let-7d stem–loop or miR-20a stem–loop were incubated with P19 nuclear extracts. Unlabeled Let-7d loop or control oligonucleotide competitors were included as indicated at 12.5, 125, or 1250 nM. Proteins were crosslinked to probes with UV light. Lin-28 was immunoprecipitated from crosslink reactions with a polyclonal antibody. Total extract (IP input) and immunoprecipitates were resolved, as indicated, on a polyacrylamide gel.

Lin-28 is necessary and sufficient for regulated Let-7 processing. (A) Pri-miRNA substrates for Let-7g and miR-17 were combined and incubated in HeLa nuclear extracts. Purified, recombinant NF-45 (control) or Lin-28 were included at 2, 20, and 200 ng per reaction. Drosha products were resolved on a denaturing polyacrylamide gel. (Arrows) Precursor products. Recombinant protein was produced in E. coli. (B) (Left lanes) Polyclonal Lin-28 antibody, or mock, was bound to protein A sepharose. P19 nuclear extracts were incubated with immobilized antibody. Resultant immunodepleted extracts were incubated with pri-miRNA substrates for Let-7g and miR-17. (Right lanes) Recombinant Lin-28 was added back to immunodepleted reactions at 100 ng/μL final concentration. Drosha products were resolved on a denaturing polyacrylamide gel. (Arrows) Precursor products. Recombinant protein was produced in HEK-293 cells.