Processing of intronic microRNAs

Abstract

The majority of human microRNA (miRNA) loci are located within intronic regions and are transcribed by RNA polymerase II as part of their hosting transcription units. The primary transcripts are cleaved by Drosha to release approximately 70 nt pre-miRNAs that are subsequently processed by Dicer to generate mature approximately 22 nt miRNAs. It is generally believed that intronic miRNAs are released by Drosha from excised introns after the splicing reaction has occurred. However, our database searches and experiments indicate that intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis. Intriguingly, cleavage of an intron by Drosha does not significantly affect the production of mature mRNA, suggesting that a continuous intron may not be required for splicing and that the exons may be tethered to each other. Hence, Drosha may cleave intronic miRNAs between the splicing commitment step and the excision step, thereby ensuring both miRNA biogenesis and protein synthesis from a single primary transcript. Our study provides a novel example of eukaryotic gene organization and RNA-processing control.

Figure 1

EST analysis of microRNA. (A) The genomic location of miRNAs relative to their hosting genes. In Supplementary Table 1, the hosting gene status for each miRNA is described in a greater detail. (B) ESTs that match EGFL7 gene. The 5′ ends of two ESTs begin at 1 or 3 nt downstream of the 3′ end of mature miR-126.

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).

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.

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.

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.

Figure 6

Splicing and cropping of four exons–three introns reporters. (A) Schematic depiction of the constructs used in this experiment. The hairpin represents the miR-26b stem–loop and the arrows indicate the position of primers. The mature miRNA sequences are written in bold whereas the mutated sequences are shown in red. (B) Northern blot analysis of miR-26b reporter expression. Two days after transfection into HEK293T cells, total RNA was extracted and analyzed by Northern blotting. (C) Time-course experiment of the reporter constructs. After transfection of the reporter plasmid into HEK293T cells, total RNA was prepared at the indicated time. The neomycin resistance gene was used as transfection efficiency control. All experiments were performed at least three times and representative results are shown.

Figure 7

A model for the processing of intronic microRNA. The exons flanking miRNA are cotranscriptionally defined and physically bound to each other by a ‘splicing commitment complex' (CC). Downstream introns may be spliced out rapidly, whereas splicing of the miRNA-harboring intron is detained. The Microprocessor cleaves the intron to release pre-miRNA that is subsequently processed by Dicer. Because the exons had already been paired and tethered to each other, splicing catalysis would still occur efficiently regardless of the discontinuity of the intron. Details are described in the text.