Gemin3 is in a complex with eIF2C2 in vivo. Immunoprecipitations were performed with monoclonal antibodies 11G9 or 12H12 against Gemin3 or with nonimmune mouse IgG from total HeLa cell lysates. The immunoprecipitates were resolved by SDS-PAGE and stained with Coomassie blue. The identity of immunoprecipitated proteins is shown on the left. Molecular mass markers (in kilodaltons) are shown on the right; (h.c) heavy chain of the antibody; (l.c) light chain of the antibody.

miRNPs sediment on sucrose gradients as ∼15S particles. Total HeLa cell lysate was sedimented on a 5%–20% sucrose gradient. Fractions were collected (indicated by numbers), resolved by SDS-PAGE, and analyzed by immunoblotting with 11G9 (anti-Gemin3), 17D10 (anti-Gemin4), and 8C7 (anti-eIF2C2). Total lane shows ∼5% of input used for gradient analysis; lane marked pellet shows ∼5% of the pellet from the gradient. (Bottom panel) fractions were pooled (indicated by brackets) and used for immunoprecipitations with 11G9. RNA was isolated, 3′-end-labeled with [5′-³²P]-pCp, and resolved by electrophoresis on a 15% denaturing polyacrylamide gel. Nucleotide size marker (M) is shown on the right. S-values are shown.

∼22-nt RNAs (miRNAs) are in a complex with Gemin3 and eIF2C2 in vivo. Immunoprecipitations were performed with 11G9 (anti-Gemin3), nonimmune mouse IgG, 8C7 (anti-eIF2C2), 2B1 (anti-SMN), and 12H12 (anti-Gemin3) from total HeLa cell lysates. RNA was isolated, 3′-end-labeled with [5′-³²P]-pCp, and resolved by electrophoresis on 15% denaturing polyacrylamide gels. Total (T) shows RNA representing ∼3% of the input used for immunoprecipitations (IPs). The lane marked M contains ³²P-labeled pBR322/MspI digest as size marker; nucleotide sizes are indicated on the left.

Gemin3 and Gemin4 associate with eIF2C2 in vivo and in vitro. (A) 8C7 recognizes a single band at ∼95 kD corresponding to eIF2C2, on Western blot of total HeLa cell lysate. Molecular mass markers (in kilodaltons) are shown on the right. (B) Immunoprecipitations (IP) were performed from total HeLa cell lysates with antibodies against Gemin3 (11G9), eIF2C2 (8C7), or nonimmune mouse IgG as negative control. The immunoprecipitates were analyzed by immunoblotting with monoclonal antibodies 8C7, 11G9, 17D10 (anti-Gemin4), 2B1 (anti-SMN), and 2E17 (anti-Gemin2) as indicated. Total (T) shows ∼3% of the input used for IPs. (*) Undissociated heavy and light chains of the antibodies used for IPs. (C,D) The indicated proteins were produced by in vitro transcription and translation in reticulocyte lysate, labeled with [³⁵S]methionine, and incubated with recombinant GST–eIF2C2 (C) or GST–Gemin3, GST–Gemin4, and GST (D); bound proteins were resolved by SDS-PAGE and visualized by fluorography. Total refers to 10% of the input fraction used for binding.

Predicted secondary structure of putative miRNA precursors and organization of miRNA gene clusters. (A) 70 nt of genomic sequence upsteam and 70 nt downstream of the cloned miRNAs were used to predict the secondary structure of putative miRNA precursors, using the computer program mfold (Mathews et al. 1999). The size of the putative miRNA precursors was then reduced to ∼80 nt, and this sequence was used again for secondary structure prediction by mfold. The putative precursor of miR-91 also contains the sequence of miR-17 on the opposite strand (colored in blue). This finding is similar to the C. elegans miR-56 and miR-56*, which are processed from the same precursor, although only miR-56 is thought to be the functional miRNA (Nelson et al. 2001). Chromosomal location is indicated. Red areas represent mature miRNAs. (B) The putative miRNA precursors are represented as boxes, and the mature miRNAs identified in this study as part of miRNPs are indicated in red. Previously reported miRNAs (Lagos-Quintana et al. 2001) not found in this study are shown in blue. The chromosomal location is indicated on the right. The miRNA cluster on chromosome 13 contains two miRNAs (miR-91 and miR-92) not previously identified; miRNA-91 and miR-17 (colored in blue) are presumably derived from the same precursor. The size of clusters in nucleotides (nt) is shown.