Structure and activity of putative intronic miRNA promoters

Abstract

MicroRNAs (miRNAs) are RNA sequences of approximately 22 nucleotides that mediate post-transcriptional regulation of specific mRNAs. miRNA sequences are dispersed throughout the genome and are classified as intergenic (between genes) or intronic (embedded into a gene). Intergenic miRNAs are expressed by their own promoter, and until recently, it was supposed that intronic miRNAs are transcribed from their host gene. Here, we performed a genomic analysis of currently known intronic miRNA regions and observed that approximately 35% of intronic miRNAs have upstream regulatory elements consistent with promoter function. Among all intronic miRNAs, 30% have associated Pol II regulatory elements, including transcription start sites, CpG islands, expression sequence tags, and conserved transcription factor binding sites, while 5% contain RNA Pol III regulatory elements (A/B box sequences). We cloned intronic regions encompassing miRNAs and their upstream Pol II (miR-107, miR-126, miR-208b, miR-548f-2, miR-569, and miR-590) or Pol III (miR-566 and miR-128-2) sequences into a promoterless plasmid, and confirmed that miRNA expression occurs independent of host gene transcription. For miR-128-2, a miRNA overexpressed in acute lymphoblastic leukemia, ChIP analysis suggests dual regulation by both intronic (Pol III) and host gene (Pol II) promoters. These data support complex regulation of intronic miRNA expression, and have relevance to disregulation in disease settings.

FIGURE 1.

Pol II intronic promoters drive intronic miRNA expression. (A) Representative diagram of the predicted Pol II intronic promoter of miR-208b. (B) Diagrams of vectors to determine functionality of Pol II intronic promoters. A genomic fragment containing the predicted promoter of miR-107, miR-206, miR-208b, miR-548f-2, miR-569, or miR-590, followed by the miRNA sequence, was cloned into a promoterless plasmid with a poly(A) signal. (C,D) Predicted intronic Pol II promoters transcribe intronic miRNAs. HEK293 cells were transfected with the constructs depicted. Active transcription from intronic promoters was detected on RNA harvested from cell lysates by RT-PCR (C) and by SYBR-green RT-qPCR (D) in the presence of Reverse Transcriptase (RT+). Nontransfected control (NTC) cells and no RT (RT−) reactions were used as controls. Relative pre-miRNA levels were normalized to β-actin. Data are mean ± SEM. (*) P < 0.05, n = 4. (E,F) HEK293 cells were transfected with plasmids containing pre-miRNA sequence with or without the predicted promoter sequence. Expression of miR-126 (E) and miR-208b (F) was determined by qPCR and was found to be significantly higher than control (endogenous) levels only in RNA obtained from cells transfected with plasmids containing the predicted promoter. Mature miRNA expression was normalized to RNU48 expression and compared with nontransfected controls. Data are mean ± SEM. (*) P < 0.05; n = 4.

FIGURE 2.

Pol III intronic promoters drive intronic miRNA expression. (A) Representative diagram of the miR-566 genomic locus in several species. (B) Diagram of gHsa-miR-566 and gMmus-Sema3F constructs. The gHsa-miR-566 construct contains the intronic sequence of SEMA3F harboring the primate-specific Alu-derived miR-566 sequence. gMmus-Sema3F construct contains the equivalent intronic sequence of Sema3F from mouse, which is devoid of intronic miRNA sequence. (C) Mir-566 expression in HEK293 cells. Mir-566 expression was detected by qPCR in HEK293 cells after transfection with gHsa-566, but not in cells transfected with gMmus-Sema3F. MiR-566 levels were normalized to 18S expression and compared with cells transfected with gMmus-Sema3F. Data are mean ± SEM. (*) P < 0.05, n = 4. (D) Mir-566 is expressed independently of Sema3F. MiR-566 and Sema3F expression were determined in HEK293 cells and PBMC cells. Data show expression of both miR-566 and Sema3F in HEK293 cells, while PBMC cells express only miR-566 and not the host gene. MiR-566 and Sema3F levels were normalized to 18S expression. Data are mean ± SEM. (*) P < 0.05, n = 4. (E–G) An intronic Pol III promoter expresses miR-128-2. (E) Diagrams of miRNA expression vectors and the Renilla luciferase reporter for assessing promoter activity of the 5′ sequence of miR-128-2. gHsa-128-2 corresponds to a genomic fragment containing pre-miR-128-2 and its upstream sequence. U6-miGFP, U6-miHD2.1, and U6-miR-128-2 are Pol III expression vectors with the U6 promoter driving miGFP, miHD2.1, or pre-miR-128-2 sequences. MiR-128-2-5′HD2.1 contains the upstream sequence of miR-128-2 as a putative promoter sequence upstream of miHD2.1. PS-HD2.1 is the Renilla luciferase reporter plasmid to test for functional miHD2.1 liberated from U6-miHD2.1 (positive control) or miR-128-2-5′-HD2.1. HSV-TKP, herpes simplex virus thymidine kinase promoter; SV40p, simian virus 40 promoter; FF Luc, Firefly luciferase; Ren Luc, Renilla luciferase; 2.1 corresponds to the target sequence of miR-HD2.1; pA, polyA; TTTTT, Pol III stop signal. (F) The predicted intronic Pol III promoter expresses miR-128-2. Data show expression of pre-miR-128-2 in HEK293 cells after transfection relative to cells transfected with negative control plasmid (U6-miGFP). RNA from striatum was used as a positive control (+Control). Pre-miR-128-2 levels were normalized to 18S expression. Data are mean ± SEM. (*) P < 0.05, n = 4. (G) The upstream sequence of miR-128-2 drives expression of miHD2.1. Luciferase assays of lysates harvested after cotransfection of PS-HD2.1 and U6-miHD2.1, U6-miR-128-2, gHsa-128-2, or miR-128-2-5′HD 2.1. Data represent mean ± SEM. (*) P < 0.05, n = 4.

FIGURE 3.

Characterization of the intronic miR-128-2 promoter by an eGFP reporter system. (A) Diagram of the eGFP-reporter constructs to differentiate Pol II and Pol III transcription. CMVeGFP contains a CMV promoter (Pol II), followed by a spacer sequence, a Pol III terminator signal, an eGFP cDNA, and a SV40pA signal. U6eGFP, pALeGFP, and miR-128-2 5′ eGFP refer to constructs harboring a U6 promoter, a Pol III Alu-derived promoter or the 5′ sequence of miR-128-2, respectively. (B) eGFP expression in HEK293 cells transfected with the eGFP-promoter reporter constructs. Scale bar, 200 μm. (C) Representative Western blot for eGFP indicates robust expression of eGFP in CMVeGFP-transfected cells, modest but detectable expression in U6eGFP-transfected cells, and no detectable eGFP expression in cells transfected with miR-128-2 5′eGFP or pALeGFP. β-actin was used as a protein-loading control. (D) RT-PCR to assess Pol III versus II transcription. HEK293 cells were transfected with the eGFP promoter-reporter constructs and RNA assayed by RT-PCR. The expected 500-bp amplicon was seen in cells transfected with CMVeGFP and U6eGFP, confirming Pol II transcription. No amplicon was observed in cells transfected with pALeGFP or miR-128-2 5′eGFP.

FIGURE 4.

Analysis of endogenous miR-128-2 promoter activity. (A,B) ChIP from ALL cells to ascertain Pol II and Pol III occupancy upstream of miR-128-2. (A) Representative PCR showing enrichment of miR-128-2 intronic sequence after ChIP with Pol II or Pol III antibodies. U6 snRNA (Pol III control) and U2 snRNA (Pol II control) are also shown. (B) Quantitation of ChIP data. Data are mean ± SEM relative to no-antibody control (n = 3). (C) Diagram showing localization of miR-128-2 within the intronic sequence of its host gene Arpp-21. (D) qPCR analysis of pre-miR-128-2 expression and its host gene in response to BDNF induction (50 ng/mL) in primary cortical neuronal cultures. Results demonstrate miR-128-2 and Arpp-21 expression increases after BDNF treatment. RNA levels of pre-miR-128-2 and Arpp-21 were normalized to β-catenin expression. Data are mean ± SEM relative to samples collected before BDNF treatment. (*) P < 0.05; Student t-test, n = 5 samples each time point.

FIGURE 5.

Hip-1 is an mRNA target of miR-128-2. (A) Cartoon depicting location of the miR-128-2 binding site in the 3′UTRs of Hip1 mRNA, as predicted and annotated by TargetScan4.1. (B) Predicted binding of miR-128-2 sequence with the Hip1 3′UTR binding site. (C) Diagram of miRNA expression vectors and Renilla luciferase reporters. U6-miGFP, U6-miHD2.1, and U6-miR-128-2 refer to Pol III expression vectors with the U6 promoter driving the expression of an artificial miRNA targeting eGFP, a miRNA harboring siRNA sequences targeting Huntingtin mRNA, or pre-miR-128-2. PS-Hip1 is a luciferase reporter construct harboring the full-length 3′UTR of Hip-1, as well as the perfect match target sequence for miHD2.1. (D) Luciferase activity of protein lysates after cotransfection of HEK293 cells with luciferase reporter constructs for Hip1 3′UTR (0.05 μg) and U6-miGFP, U6-miHD2.1, or U6-miR-128b (0.1 μg). Data are mean ± SEM. (*) P < 0.05, n = 4. (E) Representative Western blot of HEK293 cell lysate following transfection with U6 miGFP or U6 miR-128-2. β-actin was used as a protein-loading control. (F) Densitometry of westerns from three independent experiments; miGFP, 1.1 ± 0.09; miR128-2, 0.6 ± 0.013. (*) P < 0.05, n = 3. Units are density of intensity/mm².