Alternative initiation and splicing in dicer gene expression in human breast cells
© Irvin-Wilson and Chaudhuri.; licensee BioMed Central Ltd. 2005
Received: 21 December 2004
Accepted: 14 April 2005
Published: 16 May 2005
Dicer is a ribonuclease that mediates RNA interference both at the transcriptional and the post-transcriptional levels. Human dicer gene expression is regulated in different tissues. Dicer is responsible for the synthesis of microRNAs and short temporal (st)RNAs that regulate the expression of many genes. Thus, understanding the control of the expression of the dicer gene is essential for the appreciation of double-stranded (ds)RNA-mediated pathways of gene expression. Human dicer mRNA has many upstream open reading frames (uORFs) at the 5'-leader sequences (the nucleotide sequence between the 5'-end and the start codon of the major ORF), and we studied whether these elements at the 5'-leader sequences regulate the expression of the dicer gene.
We determined the 5'-leader sequences of the dicer mRNAs in human breast cells by 5'-RACE and S1-nuclease protection analysis. We have analyzed the functions of the 5'-leader variants by reporter gene expression in vitro and in vivo.
We found that the dicer transcripts in human breast cells vary in the sequence of their 5'-leader sequences, and that alternative promoter selection along with alternative splicing of the 5'-terminal exons apparently generate these variations. The breast cell has at least two predominant forms of dicer mRNAs, one of which has an additional 110 nucleotides at the 5'-end. Sequence comparison revealed that the first 80 nucleotides of these mRNA isoforms are encoded by a new exon located approximately 16 kb upstream of the reported start site. There are 30 extra nucleotides added to the previously reported exon 1. The human breast cells studied predominantly express two 5'-leader variants of dicer mRNAs, one with the exons 2 and 3 (long form) and the other without them (short form). By reporter gene expression analysis we found that the exon 2 and 3 sequences at the 5'-leader sequences are greatly inhibitory for the translation of the mRNA into protein.
Dicer gene expression in human breast cells is regulated by alternative promoter selection to alter the length and composition of the 5'-leader sequence of its mRNA. Furthermore, alternative splicing of its exon 2 and 3 sequences of their pre-mRNA creates a more translationally competent mRNA in these cells.
RNA interference (RNAi), a process of silencing gene expression, involves the generation of short, double-stranded RNA (dsRNA) molecules by an enzyme called dicer, which cleaves RNA duplexes into 21–23 base-pair oligomers [1–8]. These oligomers are called, depending on their end-point functions, small interfering RNAs (siRNA), microRNA (miRNA) or short temporal RNA (stRNA) . These small RNA molecules cause sequence-specific post-transcriptional gene silencing by guiding an endonuclease, the RNAi-induced silencing complex (RISC), to mRNA [10, 11]. This ubiquitous process has also been recently reported [12–15] in human cells to induce transcriptional silencing through promoter methylation.
Dicer gene expression is regulated in different tissues in humans . Because dicer catalyzes the biosynthesis of miRNAs and stRNAs that in turn regulate the expression of many genes, it is likely that the expression of the dicer gene itself is a highly regulated process [9–11]. While studying the published 5'-leader sequences of human dicer transcripts we noticed that it is infested with many upstream open reading frames (uORF) and out of frame AUG codons . To evaluate whether the 5'-untranslated region of the dicer transcript is in part responsible for the regulation of the dicer transcripts in human breast cells we have amplified, cloned and functionally characterized the 5'-leader sequences of human dicer transcripts from these cells. We report here that the dicer gene in breast cells is transcribed from a far upstream promoter in chromosome 14 and the sequence of the 5'-leader sequences determines the translatability of the dicer transcript and is dictated by alternative splicing of the 5'-exons.
Materials and methods
We used a series of commercially available human lines of breast cells, including human mammary epithelial (HME) cells (Clonetics, purchased through Fisher Scientific, Pittsburgh, PA, USA), MDA-MB-231, MCF-7, MDA-MB-468, MCF-10A, and BT549. We also used non-breast cells such as HeLa and HepG2. All cells, other than the HME cells, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HMEC cells were grown in medium purchased from Clonetics under their recommended conditions. Human breast carcinoma MDA-MD-231 and MDA-MD-468 cells were maintained in Leibovitz's L-15 medium supplemented with 1% antibiotic/antimycotic and 10% fetal bovine serum. MCF-10A cells were maintained in a 1:1 mixture of Ham's F12 medium, Dulbecco's modified Eagle's medium supplemented with 1% antibiotic/antimycotic, 0.098 mg/ml cholera toxin, 0.02 μg/μl epidermal growth factor, 0.5 μg/ml hydrocortisone, and 10% horse donor heard serum. BT-549 cells were maintained following standard ATCC recommended media. All cells were maintained in a humidified CO2 (5%) incubator at 37°C. Other cells were maintained and grown in ATCC recommended media and conditions [17, 18].
Complementary DNAs (cDNA) were made from total RNA (5 μg) using random primers following standard protocols [18, 19]. The cDNAs were dC-tailed and the 5'-ends of the dicer mRNAs were amplified using a dicer gene specific primer (5'-AGTTGACCAAGAACACCG-3'), and the Abridged Anchor Primer (AAP, Invitrogen, Carlsbad, CA, USA) following 5'-rapid amplification of cDNA ends (RACE) analysis protocols from Invitrogen. Amplicons were re-amplified successively using nested dicer gene specific primers (5'-TGACCAAGAACACCGTCC-3', and 5'-AAATGTCTTCCCTGAGCC-3'), and AUAP and UAP, respectively. All PCRs were done using suggested thermocycler conditions of the 5'-RACE protocol (Invitrogen). 5'-RACE products were cloned in the pCRII-Topo vector following TOPO-TA cloning protocols (Invitrogen) and the nucleotide sequences of the cloned inserts were determined by automated DNA sequencing .
S1-nuclease protection assay
Oligonucleotides that were biotinylated at the 5'-end (5'-CACAGCATGCCCAAGCTT CTGCTCTCAAAATGCTGATTCTAAGTTC-3', and 5'-GCATTTTTGTTCTAGCACAGC TTACCTTCCCACTCGCCTGCGTTTC-3') spanning the exon 2 and exon 3 boundary of the long 5'-variant and the exon 1 and exon 4 boundary of the short 5'-variant of the dicer mRNA, respectively, were custom synthesized and gel-purified (Invitrogen). The standard S1-nuclease protection protocol was followed  with the following modifications: total cellular RNA (10 μg) was co-precipitated with each probe (10 pmol) and hybridized at 65°C for 15 h. S1-nuclease (Promega, Madison, WI, USA) was used at a concentration of 500 units/ml for 90 min at 37°C. Products were analyzed in a 15% TBE-urea gel, electrophoretically transferred to Zeta Probe blotting membrane (BioRad, Hercules, CA. USA) and biotinylated protected probes were detected using the North2South HRP Detection protocol (Pierce, Rockford, IL, USA).
5'Leader sequence/Renillaluciferase constructs
The long and short form dicer 5'-leader sequences were amplified using the forward primer 5'-GCGGAAGTGGGTGTTTGTTATTTCC-3' and the reverse primer 5'-GGATCATAAACTTTCGAAGTCATTGCATTTTTGTTCTAGCACAGC-3'. Gene splicing by overlapping extension (SOE) was used to fuse dicer variants to Renilla luciferase that was amplified with forward primer 5'-GCTGTGCTAGAACAAAAATGCAATGACTTCGAAAGTTTATGATCC-3' and reverse primer 5'-CTCGAAGCGGCCGCTCTAG-3' . The Renilla luciferase ORF alone was amplified using forward primer 5'-ATGACTTCGAAAGTTTATGATCC-3' and reverse primer 5'-CTCGAAGCGGCCGCTCTAG-3'. SOEing products were then ligated into the pCRII-Topo vector (Invitrogen), digested with EcoRI (Promega) and cloned at the EcoRI site of pcDNA3.1(+) (Invitrogen) and sequenced with T7 primer to determine the orientation of the cloned insert. It is anticipated from the vector information (Invitrogen) that the Renilla luciferase transcript from these constructs will have a 133 nucleotide vector derived sequence before the 5'-leader sequences from the dicer gene at their 5'-end. Thus, the nucleotide sequence that is common to the control and the experimental transcripts is 5'-TAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCGGCTT-3'. This sequence does not have any ATG codon.
Transfection and luciferase assay
Cells were seeded at 80% confluency in a 24-well plate for 24 h in their growth media before co-transfection with one of the pcDNA3.1(+) test plasmid constructs and pGL3-control plasmid. The latter was used as a transfection normalization control. Plasmids were mixed at 0.5 μg per well and transfection was done using the Lipofectamine Plus transfection reagents (Invitrogen) using the protocol suggested by the supplier. After 20 h of incubation in complete medium at 37°C, the cells were lysed in 100 μl passive lysis buffer (Promega) and 5–20 μl of the lysate was assayed  for firefly luciferase as well as Renilla luciferase activities using Dual Luciferase Assay Reagents (Promega) following suggested protocols . Renilla luciferase activity was normalized with respect to firefly luciferase activity and presented as a ratio (relative light units). Protein contents of the extract, when needed, were determined using RC-DC reagents and protocol from BioRad Laboratories Hercules, CA, USA .
In vitrotranscription and translation
The pcDNA3.1(+) constructs containing the cloned inserts also have a T7 RNA polymerase promoter (Invitrogen). To evaluate whether there is any effect of the dicer 5'-leader sequence insert at the EcoRI site in proper orientation on the translatability of the mRNA, we used the T7 RNA polymerase/rabbit reticulocyte lysate in vitro transcription/translation system (TNT, Promega). Each plasmid DNA (1 μg) was added to 40 μl of TNT Quick Master Mix containing 1 mM of methionine (Promega). The reactions were incubated at 30°C for 75 min then cooled to 4°C. The TNT reactions were diluted 1:2 with 1x passive lysis buffer (Promega) and incubated at room temperature for 15 min. An aliquot (5–20 μl) was assayed in 100 μl of Renilla Luciferase Assay Substrate (Promega).
Each data set is presented as mean ± SEM (N = 12). Statistical significance of a difference between two series of data was tested by determining the P value . If the P value was less than 0.05, the difference was considered to be significant .
Results and discussion
Dicer mRNA has a new exon at the 5'-end
The long form of the 5'-leader sequences contains nine upstream AUG codons (Fig. 2). The short form of the 5'-leader sequence is the alternatively spliced form of the long form. In the short form exon 1 is directly joined to exon 4 and exons 2 and 3 are spliced out (Figs 2 and 3); the number of upstream AUG codons is decreased to five due to this alternative splicing (Fig. 2). Upstream AUG codons often are reported to slow down the rate of translation of an mRNA [30–37]. We tested whether the decrease in the upstream AUGs has any effect on the translatability of the dicer mRNAs (see below).
Translation of the short 5'-leader form of the dicer mRNA is more efficient
In human breast cells, the dicer gene is transcribed from a promoter that is more than 16 kbp upstream of the initiation site reported for this gene from non-breast cells . This alternative promoter selection modifies the length and composition of the 5'-leader sequences of its mRNA. Furthermore, alternative splicing of the exon 2 and 3 sequences of its pre-mRNA creates a more translationally competent mRNA in these cells. Breast cell dicer mRNAs have a high number of upstream AUG codons with in frame stop codons. Translational efficiency and/or stability of the RNA are decreased due to this type of 5'-leader sequence [34–37]. The non-sense mediated degradation pathway of mRNA decay may also be stimulated by these uORFs [38, 39]. It is interesting to note that there is an association between active dicer mRNA expression and the invasiveness of the breast cell line. Recently we found that the zinc finger repressor protein SLUG may have the determinative role in the invasiveness of the breast cell [40, 41]. We are exploring whether dicer regulates SLUG gene expression in human breast cells. The mechanisms by which the 5'-leader sequences of the dicer mRNAs in breast cells regulate the expression of this gene are yet to be determined.
American Type Culture Collection
human mammary epithelial
polymerase chain reaction
rapid amplification of cDNA ends
RNAi-induced silencing complex
small interfering RNA
splicing by overlapping extension
short temporal RNA
upstream open reading frame.
Supported by the MMC/VICC cancer partnership grant #1U54CA091408-010003 from NCI and the DOD grant #DAMD17-00-1-0341 to GC and graduate training fellowship #1T32GM062758-01 from NIH to CVIW.
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