Processing of miRNA-harboring introns

In order to examine whether the miRNA hairpin is indeed released from an unexcised intron, we depleted Drosha and determined the level of the unspliced transcripts. If Drosha cleaves unexcised introns, it is expected that the unspliced introns would accumulate in Drosha-depleted cells. SiRNA against Drosha was transfected into HeLa cells and total RNA was analyzed by reverse transcription–PCR (RT–PCR). The primer sets had been designed to amplify both the nascent transcripts and the partially spliced transcripts (Figure 2A, left panel). Structures of the genes analyzed in this study are shown in Supplementary Figure 1. The first set of primers (Figure 2A, Puf and Pur, shown in red) amplifies the upstream region. Two distinct transcripts (Un and Up, shown in red) were detected using this upstream primer set. The longer PCR product (Un) amplified by Puf and Pur corresponds to a nascent transcript retaining the upstream intron. The shorter PCR product (Up) corresponds to the partially spliced transcript that lacks the upstream intron. The second set of primers (Figure 2A, Pdf and Pdr, shown in blue) amplifies the downstream region. These primers also detected two kinds of transcripts: nascent (Dn) and partially spliced transcripts (Dp) (Figure 2A, shown in blue). In all of the cases that we analyzed, partially spliced transcripts (Up and Dp) accumulated upon Drosha depletion, whereas the levels of nascent transcripts (Un and Dn) only slightly increased (Figure 2A, right panel).

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Figure 2

Splicing of microRNA-encoding introns. (A) RT–PCR for the detection of nascent and partially spliced transcripts. The positions of primers (arrows), exons (filled boxes) and introns (solid lines) are indicated. PCR products are depicted as either red or blue solid lines. SiRNA duplexes targeting luciferase (siLuc) or Drosha (siDro) were transfected into HeLa cells and, after 48 h, total RNA was prepared and used for RT–PCR. (B) Real-time PCR of the nascent or partially spliced transcript of MCM7 gene. Different RT primers were used to distinguish these two transcripts, and the same PCR primer set was used for real-time PCR reaction (n=5).

We also carried out real-time PCR experiments to confirm the above results. Two different RT primers were used to distinguish nascent and partially spliced transcripts. The RT primer that is complementary to the downstream intronic sequence was to detect the nascent transcript (Figure 2B, RT primer 1), whereas the RT primer that anneals to the downstream exon–exon junction could detect the partially spliced transcript (Figure 2B, RT primer 2). Consistent with the above results, only the partially spliced transcripts accumulated upon depletion of Drosha. This indicates that Drosha is required specifically for the turnover of the partially spliced transcripts. There may be at least two explanations to this observation. The first and obvious possibility is that, in Drosha-depleted cells, the partially spliced transcripts accumulated because cropping reaction was slowed down. In a normal condition, Drosha would rapidly remove these transcripts by cleaving away the hairpin in the intron. Second possibility is that Drosha may somehow be required for the splicing of miRNA-containing introns but not of the adjacent introns. The depletion of Drosha may have slowed down the splicing of miRNA-containing introns, resulting in the accumulation of transcripts that retain the miRNA-harboring introns.

To test this possibility, we determined the amount of spliced mRNA in Drosha-depleted cells by RT–PCR (Figure 3). If Drosha is indeed required for splicing, splicing products (mature mRNA) would be reduced in Drosha-depleted cells. Primers that are complementary to the neighboring exons were used to detect spliced mRNAs (Figure 3A). For all the genes that we tested (CTDSP1 containing miR-26b, MCM7 containing miR-106b cluster, EGFL7 containing miR-126 and EML2 containing miR-330), the levels of fully spliced mRNAs remained unaffected in Drosha-depleted cells compared to those in control siRNA-treated cells. We also performed quantitative real-time PCR on MCM7 mRNA (Figure 3B). The MCM7 gene contains miR-106b cluster in its 13th intron. As expected, the level of spliced mRNA in Drosha-depleted HeLa cells was not significantly different from that in control siRNA-treated cells. Therefore, it is unlikely that Drosha is required for the splicing of the miRNA-harboring introns.

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Figure 3

The production of spliced mRNA is unaffected by miRNA cropping. (A) RT–PCR to detect spliced mRNA. SiRNA duplexes targeting luciferase (siLuc) or Drosha (siDro) were transfected into HeLa cells and, after 48 h, total RNA was prepared and used for RT–PCR. (B) Quantitative real-time PCR of MCM7 spliced mRNA. The arrows and the black bar with an asterisk indicate the position of PCR primers and Taqman probe, respectively. Each C_t value of MCM7 was normalized to the C_t value of GAPDH (n=6). (C) Depletion of DGCR8 exerts the same effects as those of Drosha on the processing of miRNA-encoding introns. SiRNA was transfected into HeLa cells as in panel A and Figure 2A.

Comparable results were observed when DGCR8, the essential cofactor for Drosha, was depleted instead of Drosha (Figure 3C). As in Drosha-depleted cells, the partially spliced transcript retaining miRNA-encoding intron (Up and Dp) accumulated, whereas the spliced mRNA level did not significantly change in DGCR8-depleted cells. On the contrary, the knockdown of other genes that are not involved in cropping (exportin-5, DDX5 and EWSR) did not alter the level of these transcripts (Figure 3C).

To generalize our conclusion, we randomly selected several TUs and determined the levels of unspliced transcripts and spliced mRNAs by RT–PCR (Figure 4). In all cases that we tested, the unspliced transcripts accumulated in Drosha-depleted cells, whereas the level of spliced mRNA remained largely unchanged. The positions of these miRNAs inside the corresponding introns are highly variable. Also, the lengths of the introns vary greatly (ranging between ~2000 and ~10 000 nt). Thus, the processing of miRNA-harboring introns is unlikely to be affected by the location of miRNA or the length of the intron.

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Figure 4

Processing of miRNA-harboring introns is unaffected by the length of the intron and the location of miRNA. The black triangle indicates the location of miRNA.

Splicing is not a prerequisite for cropping

In order to examine if the pre-mRNA splicing and cropping process have any effects on each other, we designed reporter constructs that contain the sequences of the BCL2L2 gene under the control of the human cytomegalovirus promoter (Figure 5A). The RNA transcribed from this construct is expected to contain two exons and one intron. A let-7a-1 miRNA hairpin was inserted into the middle of the intron so as to determine the effects of having an miRNA hairpin upon splicing (Figure 5A, BCL-let-7a). In addition, a splicing-deficient construct was designed by introducing mutations at the 5′ and 3′ consensus splice sites to examine the influence of splicesome assembly on the cropping process (Figure 5A, BCL-m-let-7a). After transfection of these plasmids into HEK293T cells, total RNA was extracted and analyzed by RT–PCR. As controls, the transcripts for GAPDH and the neomycin resistance gene were measured (Figure 5B). The neomycin resistance gene is encoded in the reporter plasmid; so the expression of this gene is expected to reflect the transfection efficiency.

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Figure 5

Pre-mRNA splicing is not required for cropping. (A) Reporter constructs. Upper row: wild-type BCL2L2 mRNA (exon: filled box; intron: solid line). The miRNA stem–loop was inserted into the EcoRI site in the intron. Middle row: miRNA-inserted construct. Lower row: splicing-deficient form of miRNA-inserted construct (red dots indicate the sites of mutation). The mutated sequences are indicated in red. (B, C) RT–PCR and Northern blotting. Reporter constructs were transfected into HEK293T cells and, 2 days later, total RNA was extracted for RT–PCR and Northern analysis. In panel C, the band indicated with an asterisk (^*) represents other members of the endogenous let-7a family. 18S rRNA stained with EtBr is shown as loading control.

The insertion of the let-7a-1 hairpin did not notably alter the levels of mature BCL2L2 mRNA (Figure 5B, primers Pf and Pr, lanes 2 and 3), which again demonstrates that cropping of an intronic miRNA hairpin does not significantly affect the splicing efficiency of the given intron. The pre-mRNA with mutated splice sites (BCL-m-let-7a) remained unspliced as expected (Figure 5B, lane 4).

The levels of let-7a miRNA expressed from these reporter constructs were determined by Northern blotting (Figure 5C). Let-7a miRNA was successfully expressed from the reporter constructs (Figure 5C, lanes 3 and 4). Importantly, the level of pre-let-7a-1 from the splicing-deficient construct (BCL-m-let-7a) was only slightly decreased compared with the wild-type construct (BCL-let-7a) (Figure 5C, compare lanes 3 and 4). These results demonstrate that a splicing event is not a prerequisite for pri-miRNA processing.

EST analysis

A BLAT search (Kent, 2002) was initially carried out to identify the position of the miRNA locus. After identifying the miRNA site using BLAT, its position was compared with gene loci using RefSeq, Known gene, human mRNA and human EST database. When each miRNA was matched with a gene that was verified as having protein-coding capabilities, the hosting gene status was marked with ‘Known gene' (see Supplementary Table 1). If the miRNA species was matched to a full-length mRNA, but not matched to known gene or with RefSeq, that is, its protein-coding capability was only predicted, a ‘Predicted' hosting gene status was assigned. Finally, when the miRNA was matched only with an EST, the hosting gene status was designated as ‘EST only'. We used spliced ESTs only, as their unspliced counterparts cannot distinguish whether the sequence is exonic or intronic.

The cleavage products of Drosha were analyzed using all types of EST, both spliced and unspliced EST. If either the 5′ or 3′ ends of the mature miRNA duplex were not known exactly, the Drosha and Dicer cleavage sites were estimated based on the assumption that all miRNA duplexes contain 2-nt 3′ overhangs at both ends.

Cell culture and transfection

HeLa and HEK293T cells were cultured in DMEM (WelGENE) supplemented with 10% FBS (WelGENE). Transfection to HEK293T cells was carried out using the calcium-phosphate method. Lipofectamine 2000 (Invitrogen) was used for transfection of HeLa cells.

Plasmid construction

The BCL2L2 and CTDSP1 genes were PCR-amplified using HeLa genomic DNA. The primers for the BCL2L2 reporter were 5′-GAA TTC TGA TGG CGA CCC CAG CCT CGG CC-3′ (forward) and 5′-GCG GCC GCT CAC TTG CTA GCA AAA AAG GCC C-3′ (reverse). The primers for the miR-26b reporter were 5′-GAC CCC AGT CCA ATA CCT GCT-3′ (forward) and 5′-AGC ATT GTC TGG ATG GAA GAC-3′ (reverse). The PCR product was inserted into the pGEM-T easy vector (Promega).

In addition, the let-7a-1 miRNA sequence was amplified by PCR, cloned into pGEM-T easy and verified by sequencing. The primers for let-7a-1 cloning were 5′-GAT TCC TTT TCA CCA TTC ACC CTG GAT GTT-3′ (forward) and 5′-TTT CTA TCA GAC CGC CTG GAT GCA GAC TTT-3′ (reverse). The BCL2L2 gene was subcloned into a FLAG-tagged-pcDNA3 vector (Invitrogen). And the miRNA sequence was then inserted at the EcoRI site in the intron of BCL2L2.

Site-directed mutagenesis was carried out using the QuickChange Site-Directed Mutagenesis kit (Stratagene) to generate the splicing-deficient construct. The primer sets were 5′-AGT GGG GGC TGG GTA CAA AGC TTC TCA ATT GC-3′ (forward) and 5′-GCA ATT GAG AAG CTT TGT ACC CAG CCC CCA CT-3′ (reverse) for the 5′ intronic consensus sequence. The primers used for the 3′ intronic consensus sequence were 5′-TCC CTT CTC TCC CAC CTG CGG AGT TCA CAG CT-3′ (forward) and 5′-AGC TGT GAA CTC CGC AGG TGG GAG AGA AGG GA-3′ (reverse). The cropping-deficient mutation of miR-26b stem–loop was generated by site-directed mutagenesis using the following primers. Forward:

5′-GCC TGT TCT CCA TTA GAC TGC TCG GGG ACC GGT G-3′, and reverse: 5′-CAC CGG TCC CCG AGC AGT CTA ATG GAG AAC AGG C-3′.

RNA interference

SiRNA duplex was transfected into HeLa cell using RNAiFect transfection reagent (Qiagen) according to the manufacturer's instructions (Lee et al, 2003; Han et al, 2004).

RT–PCR

RT was carried out using the Superscript II kit (Invitrogen) according to the manufacturer's protocol. Total mRNA was prepared using Trizol (Invitrogen), treated with DNase I (Takara) and then reverse-transcribed using an oligo-dT primer (Invitrogen). After RT, the RNA was digested using RNase A (Ambion). The primers used in these analyses were miR-26a-2_exon_forward:

5′-ATC AAC AAT GCT GAC TTC ATA GTG-3′,

miR-26a-2_exon_reverse: 5′-CTT GGC CAG GCT GGC AGT GAA GAG-3′,

miR-26a-2_intron_reverse: 5′-CTT GGC CAG GCT GGC AGT GAA GAG-3′,

miR-26b_intron_forward (Pdf): 5′-CCG GGA CCC AGT TCA AGT AAT TCA-3′,

miR-26b_intron_reverse (Pur): 5′-TCA GCC TAG TGC AGG AAT CCA G-3′,

miR-26b_exon_forward: 5′-AGT GAA CAA CGC GGA CTT CAT C-3′,

miR-26b_exon_reverse: 5′-CTT GGC GAG GCT AGC AGT GAA CAG-3′,

miR-26b_upexon_forward (Puf): 5′-AAG ATC TGC GTG GTC ATC GAC-3′,

miR-26b-downexon_reverse (Pdr): 5′-TGT CCA GCA GGT CAG CTA CTG-3′,

miR-126_exon_forward: 5′-CAG TTA CTG GTG CCA GTG TTG GGA-3′,

miR-126_exon_reverse: 5′-CAC CCT GGA CTG CAG CCT CTG-3′,

miR-126_intron_forward (Pdf):

5′-CAG CGC AGC ATT CTG GAA GAC-3′,

miR-126_downexon_reverse (Pdr): 5′-GAG CTG CTG GAA GGA GTG CAC-3′,

miR-330_intron_forward (Pdf): 5′-GGT GAC TCC CTT CTT CCA GGA TCG-3′,

miR-330_exon_forward: 5′-CCA CCC CCG GCC CCG AAG CTC CGC-3′,

miR-330_exon_reverse: 5′-CAC TGA AGA TAA CTT CTT TGG-3′,

miR-330_downexon_reverse (Pdr):

5′-GAG CGT GTG TCC AGG CTG TAG-3′,

miR-106b-93-25_upexon_forward (Puf): 5′-TTG AAC CTC TGG ACA TGA AGC TC-3′,

miR-106b-93-25_downexon_reverse (Pdr): 5′-TAT CAC ATC TGC TGG TCT CTG AGT C-3′,

miR-106b-93-25_intron_forward (Pdf): 5′-CCT CAC AGG ACA GCT GAA CTC C-3′,

miR-106b-93-25_intron_reverse (Pur): 5′-ACT CAC AAA ACA GGA GTG GAA TCC-3′,

miR-106b-93-25_exon_forward: 5′-GCA TAC GTG GAG ATG AGG CGA G-3′,

miR-106b-93-25_exon_reverse: 5′-CCC CTT GTC TCC TAG AAG AGA G-3′,

miR-15b-16-2_intron_reverse: 5′-TTT CAG TAG TAG TGG TTC CAC CAA G-3′,

miR-15b-16-2_exon_forward: 5′-ATG TAT CCA GAA CGG CCT GCA-3′,

miR-15b-16-2_exon_reverse: 5′-CAT ACC CTC ATC GTG TTC AGT CTG-3′,

miR-26a-1_intron_reverse: 5′-CTT TGC CCA CAA GAC TCC TCG T-3′,

miR-26a-1_exon_forward: 5′-CGG TTG AAA TCG ATG GAA CTA TAC A-3′,

miR-26a-1_exon_reverse: 5′-AAG CTG GCA GTA AAG AGC ACA CAT-3′,

miR-33_intron_reverse: 5′-CGT AGA CCA GGT GAT GCT CCA C-3′,

miR-33_exon_forward: 5′-GTT CCT TCT GCC ATT GCG AGA G-3′,

miR-33_exon_reverse: 5′-GCT GTC CGT AGC GAC AGT AGC AG-3′,

miR-98_intron_reverse: 5′-ACA TCT GCC TCA CCC TAA ACA CC-3′,

miR-98_exon_forward: 5′-GAC TCC ACT GAA CAG AAT CTC TCA G-3′,

miR-98_exon_reverse: 5′-TGC TGT TGT GAG GAT TGC AGA GT-3′,

miR-101-2_intron_reverse: 5′-TGC TTA TCA GTC TTC CCT CTC TGC-3′,

miR-101-2_exon_forward: 5′-TGA CCC TTG GAC AGC AGG ATG-3′,

miR-101-2_exon_reverse: 5′-ATG CCA ACA CAG GTC ATC AGC A-3′,

miR-107_intron_reverse: 5′-CAA CTT TGT ATG TCA CCA GCT CCA C-3′,

miR-107_exon_forward: 5′-CGA GAT TCC ATC AGC AAG GAA GA-3′,

miR-107_exon_reverse: 5′-TAT GCC AGC AGC TTC ATG GAG A,

BCL2L2_Pf: 5′-GAA TTC TGA TGG CGA CCC CAG CCT CGG CC-3′,

BCL2L2_Pr: 5′-GCG GCC GCT CAC TTG CTA GCA AAA AAG GCC C-3′, neomycin-resistance gene: forward: 5′-ATG ATT GAA CAA GAT GGA TTG CAC-3′, and reverse: 5′-TCA GAA GAA CTC GTC AAG AAG GC-3′.

Real-time PCR

Real-time PCR was conducted using the comparative C_t method with the Applied Biosystems 7300 Real-Time PCR system. The primers used for RT were 5′-TTC ACT AAA CCA CTC ACC TAG-3′ (for nascent mRNA, RT primer 1) and 5′-TCT CTG AGT CCT AGC TGT CT-3′ (for partially spliced mRNA, RT primer 2). Real-time PCR reaction was conducted by using the SyBR green method, and the PCR primers were 5′-CCT CAC AGG ACA GCT GAA CTC C-3′ (forward) and 5′-CCC CTT GTC TCC TAG AAG AGA G-3′ (reverse).

To measure the level of the MCM7 spliced mRNA, the TaqMan probe was designed to bind the splice junction, and primers were designed to anneal at adjacent positions to the probe. The probe sequence is 5′-6FAM-TGC TCT GGC ACG TCT GAG AAT GGT G-TAMRA-3′. The PCR primers were 5′-GCT ATC CTG CGC CTT TCC A-3′ (forward) and 5′-TCA CAT CTT CTT TCT CCA CCA CAT-3′ (reverse). The TaqMan GAPDH endogenous control kit was purchased from Applied Biosystems.

We thank Dr Dong-Ki Lee, Sun Woo Hong, and Yoontae Lee for critical comments and technical help, Drs David Bartel and Christopher Burge for discussions on splicing and SeungYoon Nam and Dr Sang Hyuk Lee for their advice on database searches. We are also grateful to Dr Sang Hyun Park, Sung Hee Baek, Deog Su Hwang and to the members of our laboratory for their assistance with designing these experiments, and YKK thanks The Bioneers, the research group of undergraduates at Seoul National University. This work was supported by grants from the National Research Laboratory Program (M1050000010905J000010910), the Molecular and Cellular BioDiscovery Research Program (2004-01749), the Korean Research Foundation Grant (KRF-2005-005-J16001) and the BK21 Research Studentship.