Large RNAs from the EEmiRC locus. (A–C) Northern analysis of total RNA from ES cells grown on feeders (lane 1), ES cells grown without feeders (lane 2), ES cells differentiated with retinoic acid in monolayer (lane 3), and ES cells differentiated into embryoid bodies without (lane 4) and with (lane 5) retinoic acid. Hybridization was performed with a random primed DNA probe (A) or with single-stranded RNA probes antisense (B) or sense (C) to the mature microRNAs. The β-actin loading control is given in D. (E) Northern analysis of the large EEmiRC RNA intermediates in ES (lane 1) and TS (lane 2) cells is shown in the top panel. The β-actin mRNA (bottom panel) serves as a loading control. The corresponding analyses of the mature miR-292-s, miR-292-as, miR-294, and the tRNA-Ile-ATT loading control are shown in F. (G) Subcellular localization of the large EEmiRC RNAs (top panel) or the GAPDH mRNA control (bottom panel). Northern analysis of total (lane 1), cytoplasmic (lane 2), and nuclear (lane 3) RNA was performed as in A. The apparent change in the mobility of band A RNA in lanes 1 and 3 is due to differences in the total RNA amounts loaded. (H) Short RNA Northern analysis of the subcellular localization of mature miR-292-as (top), tRNA-Ile-ATT (middle), and U6 snRNA (bottom). Total, cytoplasmic, and nuclear RNAs are analyzed in lanes 1,2,3, respectively.

Effects of Drosha knockdown on EEmiRC RNA expression. (A) ES cells were transfected with a Drosha siRNA (even lanes) or an EGFP control siRNA (odd lanes). Large EEmiRC RNAs (top panel) were monitored by Northern analysis at 24, 48, and 72 h post-transfection. The positions of bands A–D are indicated. The position of an additional RNA that was up-regulated in response to Drosha knockdown is shown by an asterisk. The β-actin loading control is given in the bottom panel. (B) ES cells were transfected twice with an EGFP siRNA (lane 1) or a Drosha siRNA (lane 2) and the large EEmiRC RNAs were monitored by Northern analysis at 6 d after the first transfection (top panel). The β-actin loading control is shown in the bottom panel. (C) Drosha mRNA levels (top panel) in cells transfected with an EGFP siRNA (lanes 1,3) or a Drosha siRNA (lanes 2,4) monitored by RT-PCR at 48 or 72 h post-transfection. Amplification of the β-actin mRNA is shown in the bottom panel. No reverse transcriptase was added to the reactions analyzed in lanes 5. Lanes 1–5 contained 1 μg total RNA. The titration of 1, 0.1, and 0.01 μg total RNA in lanes 6–8 shows that the assay is in the linear range. (D,E) Short RNA Northern analyses of miR-292-as and pre-miR-292-as (top panels) or tRNA-Ile-ATT (bottom panels) in the experiments shown in panels A and B, respectively.

Splicing of the EEmiRC primary transcript and Drosha processing at pre-miR-290. (A) The positions of the RNAse protection probes (rpA and rpB) superimposed onto the schematic representation of the EEmiRC locus as in Figure 1. (B,C) Total RNA from ES cells transfected with an EGFP control siRNA (lane 2) or a Drosha siRNA (lane 3) was hybridized with probe rpA (B) or rpB (C) and subjected to RNAse protection analysis. The molecular weight marker is shown in lane 1. (D) The RT-PCR products obtained from total ES cell RNA with probes flanking the predicted intron are shown in lane 3. The reaction analyzed in lane 2 did not contain reverse transcriptase. The molecular weight marker and PCR of the genomic EEmiRC BAC are shown in lanes 1 and 4, respectively. (E) Positions of the splice sites deduced from D. Exon sequences are given in capital letters; the intron is in lowercase. The polypyrimidine tract is underlined.

Bioinformatic analysis of EEmiRC. (A) Schematic representation of the murine, human, canine, and bovine EEmiRC loci. Sequence features are given according to the legend. (B) Multiple sequence alignment of the murine (experimentally identified miR-290–295 and predicted mm-X), human (experimentally identified miR-371–373), canine (predicted cf-A–C), and bovine (predicted bt-A,B) pre-miRNA hairpins. The positions of cloned mature miRNAs and the two strands of the hairpin stem are underlined. (C) Multiple sequence alignment of the four conserved EEmiRC core promoter sequences.

Mapping of the large EEmiRC RNAs. (A) Mapping of the EEmiRC RNAs with oligonucleotide probes. Northern hybridizations with oligonucleotide probes (Table 1) corresponding to the indicated positions within the EEmiRC locus are shown. In each individual panel, the left lane contains total RNA from ES cells and the right lane contains total RNA from NIH/3T3 cells. Hybridization with a random primed probe and the positions of the four major RNA species are given on the left. Robust detection of band A can only be achieved after overloading the membranes (top panels). The EEmiRC locus representation is according to Figure 1A and includes a schematic of the intron preceding pre-miR-290. (B) Schematic representation of the RNA species deduced from A. (C) Polyadenylation of the EEmiRC transcripts. Northern analysis of total ES cell RNA (lane 1), ES cell RNA not bound to oligo(dT) cellulose (lane 2), and RNA bound to oligo(dT) cellulose (lane 3). Analysis of the EEmiRC transcripts and the polyadenylated GAPDH mRNA control are shown in the top and bottom panels, respectively. (D) Mapping of the 5′ end of the EEmiRC pri-miRNA. Final PCR amplification of the 5′ RACE products from NIH/3T3 (lanes 3,4) or ES (lanes 5,6) cell total RNA without (lanes 3,5) or with (lanes 4,6) TAP treatment. No 5′ RACE template was added to the PCR reaction shown in lane 2. The molecular weight marker is shown in lane 1. (E) The transcription start site (asterisk, +1) determined from the experiment shown in D is superimposed onto the alignment of the putative mouse (MM) and human (HS) EEmiRC promoter regions. The box highlights the conserved TATA element.

Expression of EEmiRC in transient transfections. (A) Maps of the transfected constructs. pEEmiRC contains a genomic fragment spanning positions −2003 to +4893 relative to the EEmiRC transcription start site. In pEEmiRC::EGFP positions +790 to +3017 are replaced by the EGFP coding sequence. In plasmids pEEmiRC(ΔTATA) and pEEmiR-C::EGFP(ΔTATA) positions −199 to +29 have been deleted. In pCMV-EEmiRC a fragment spanning the region from +34 to +3082 is placed between the cytomegalovirus immediate early promoter and the bovine growth hormone polyadenylation signal. (B) EGFP expression in ES cells monitored by FACS. Cells were transfected with pEEmiRC::EGFP (green traces, superposition of three independent transfections) or pEEmiRC::EGFP(ΔTATA) (red traces, superposition of three independent transfections). The blue trace shows the background fluorescence of cells transfected with a LacZ expressing construct. (C) Transfection of HEK-293 cells as in B. (D) Northern analysis of the expression of large EEmiRC RNAs (top panel) or EGFP mRNA (middle panel) in HEK-293 cells transfected with a LacZ expressing construct, pEEmiRC, pEE-miRC(ΔTATA), pEEmiRC::EGFP, pEEmiRC::EGFP(ΔTATA), or pCMV-EEmiRC (lanes 2–7, respectively). RNA from untransfected ES cells is shown in lane 1. Lane 8 is a shorter exposure of lane 7. The β-actin loading control is given in the bottom panel. (E) The levels of miR-292-as and pre-miR-292-as (top panel) and the tRNA-Ile-ATT loading control (bottom panel) in the samples from D are monitored by short RNA Northern analysis.