Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping

Abstract

The ancient MYH7b gene, expressed in striated muscle and brain, encodes a sarcomeric myosin and the intronic microRNA miR-499. We find that skipping of an exon introduces a premature termination codon in the transcript that downregulates MYH7b protein production without affecting microRNA expression. Among other genes, endogenous miR-499 targets the 3' untranslated region of the transcription factor Sox6, which in turn acts as a repressor of MYH7b transcriptional activity. Thus, concerted transcription and alternative splicing uncouple the level of expression of MYH7b and miR-499 when their coexpression is not required.

FIG. 1.

MYH7b and miR-499 coexpression profile. (A) MYH7b mRNA levels were assayed by real-time PCR performed on cDNA prepared from mouse embryos at 6.5 and 8.5 days postcoitum (dpc) (top). Reactions were normalized to the 18S rRNA. Expression of miR-499, graphed as the fold change in expression from embryonic time point 6.5 dpc to 8.5 dpc (bottom). Quantification of miR-499 was determined using the comparative C_T method from miRNA TaqMan real-time PCR, normalized to snoRNA202. (B) MYH7b mRNA levels were assayed by real-time PCR performed on cDNA prepared from 12-week-old adult mouse brain (B), quadriceps (Q), tibialis anterior (TA), gastrocnemius (G), ventricle (V), diaphragm (D), and soleus (S) (top). Reactions were normalized to the 18S rRNA. miR-499 Northern blot quantification was carried out on the same RNA used for the MYH7b real-time PCR experiments (bottom). U6 snRNA was used as a loading control. Error bars indicate the standard errors of the means calculated from four animals.

FIG. 2.

Regulation of MYH7b and miR-499 expression. (A) Effect of thyroid hormone on the expression of MYH7b and miR-499 in 12-week-old mouse heart. Gene and miRNA expression levels were measured under normal euthyroid (Eu) hormone levels, a PTU-induced hypothyroid condition (PTU), and a hyperthyroid condition due to 2 days of T₃ injection post-PTU ingestion (PTU+T3). Total MYH7b mRNA transcripts in the heart were quantified by real-time PCR and normalized to the 18S rRNA (top). miR-499 Northern blot quantification was carried out on total RNA from the hearts of mice from each thyroid condition, normalized to U6 snRNA (bottom). Error bars indicate the standard errors of the means calculated from four animals. (B) Effect of 48 h of phenylephrine (PE) treatment (20 μM) on the expression of MYH7b and miR-499 in neonatal rat ventricular myocytes. MYH7b expression was measured by quantitative real-time PCR and normalized to 18S rRNA (top). miR-499 expression was quantified by miRNA TaqMan Real-time PCR, graphed as the fold change with PE treatment compared to control levels of miR-499 normalized to snoRNA202 (bottom). Error bars indicate the standard errors of the means calculated from four independent experiments.

FIG. 3.

Comparison of MYH7b and MYH-β gene activity during C2C12 myoblast differentiation. (A) MYH7b and MYH-β mRNA levels were determined by semiquantitative RT-PCR analysis. RNA was isolated from C2C12 cells before differentiation (day 0) through 4 days postdifferentiation (days 1 to 4). PCRs carried out for 25 cycles (25c) and 30 cycles (30c) were resolved on a 2% agarose gel and visualized after ethidium bromide staining (top). Expression levels of the intronic miRNAs, miR-499 derived from MYH7b and miR-208b derived from MYH-β, were determined by Northern blot analysis on the same RNAs used for the RT-PCR analysis (bottom). MYH7b mRNA was translated and found in preparations of myofibril-type proteins. (B) Sarcomeric proteins purified from mouse soleus muscles through differential solubility and centrifugation were separated by SDS-PAGE and detected by silver staining. Purified fractions were then subjected to mass spectrometry analysis. MyHCs, myosin heavy chains; MyBP-C, myosin binding protein C; MyLCs, myosin light chains.

FIG. 4.

Sequence analysis of MYH7b exonic and intronic sequences. (A) Comparison of intronic sequences (∼100 nucleotides) bordering MYH7b exon 7 across species. The red box indicates the intronic U-rich motif located downstream of MYH7b exon 7. (B) Comparison of exon 7 nucleotide sequence conservation across species for MYH7b (94% identity) and MYH-β (78% identity). (C) Comparison of nucleotide sequence conservation across species for MYH7b exon 9 (79.6% identity) and the corresponding MYH-β exon 8 (78.5% identity). (D) Comparison between MYH7b and MYH-β protein sequences and codon usage in the highly conserved GESGAGK region of exon 7. Protein sequence is conserved to maintain the required GESGAGK motif, but alternate codon usage between the two genes results in differences in nucleotide sequence. In all panels, conserved nucleotides and amino acids are indicated by blue highlighting.

FIG. 5.

Alternative splicing of MYH7b exon 7. (A) RT-PCR analysis of RNA isolated from 12-week-old adult mouse brain (B), quadriceps (Q), tibialis anterior (TA), gastrocnemius (G), ventricle (V), atria (A), diaphragm (D), and soleus (S) (top). Reactions carried out with Cy5-labeled primers located in MYH7b exons 5 and 9 were separated by 8% native PAGE and subsequently quantified by phosphorimager analysis. Mobility of the PCR bands corresponding to splicing products that either include or skip exon 7 is indicated. The percentage of exon 7 inclusion or skipping was calculated from tissues of four different mice (bottom). Error bars indicate the standard errors of the means. (B) RT-PCR analysis of human RNA isolated from heart (H), skeletal muscle (S) and brain (B) and RNA isolated from zebrafish (Z) was performed with primers located in MYH7b exons 5 and 9 (top). Reaction mixtures containing Cy5-labeled primers and product quantifications were carried out as described for panel A. Zebrafish products were resolved by 2% agarose gel and visualized after ethidium bromide staining. Each PCR product was purified and sequenced. Asterisks indicate transcripts with skipped exon 7 and activation of a cryptic 5′ splice site downstream of exon 5. Mobility of the PCR bands corresponding to splicing products that either include or skip exon 7 is indicated. The percentage of exon 7 inclusion or skipping in heart, skeletal muscle, and brain, was calculated from several independent experiments (bottom).

FIG. 6.

Comparison of MYH7b and MYH-β exon 7 splicing during C2C12 myoblast differentiation. (A) Splicing of exon 7 of MYH7b and MYH-β was monitored in C2C12 cells before differentiation (day 0) through 4 days postdifferentiation (days 1 to 4) with Cy5-labeled primers, as described in the legend of Fig. 5. Mobility of the PCR bands corresponding to splicing products that either include or skip exon 7 is indicated. The PCR product derived from skipping of MYH-β exon 7 was not detected. A comparison of MYH7b and MYH-β promoter activity during C2C12 myoblast differentiation is shown. (B) The 1-kb promoter regions of MYH7b (left) and MYH-β (right) were cloned in the luciferase reporter plasmid pGL3. Constructs were transfected into C2C12 myoblasts with Lipofectamine 2000, and luciferase activity was measured before differentiation (day 0) through 4 days postdifferentiation (days 1 to 4). Firefly luciferase activity was normalized to Renilla luciferase activity. Error bars indicate the standard errors of the means calculated from four independent experiments.

FIG. 7.

Cycloheximide treatment of C2C12 cells increases the level of MYH7b mRNA carrying a PTC induced by exon 7 skipping. At 2 days postdifferentiation, C2C12 myotubes were exposed to cycloheximide (100 μg/ml) for 2 h. (A) Total MYH7b and MYH-β mRNA levels were quantified from untreated (C) or cycloheximide-treated (CHX) C2C12 myotubes by real-time PCR as described in the legend to Fig. 1. Error bars indicate the standard errors of the means calculated from three independent samples (MYH7b, P < 0.05; MYH-β, P = 0.69). (B) miR-499 and miR-208b Northern blot quantifications were carried out on the same RNA used in the experiment shown in panel A. Expression levels of miR-499 and miR-208b were quantified from untreated or cycloheximide-treated C2C12 myotubes as described in the legend of Fig. 1. Cycloheximide treatment did not result in any significant change in either miR-499 or miR-208b expression. Error bars indicate the standard errors of the means calculated from three independent samples. (C) Representative RT-PCR (27 cycles) from RNA isolated from untreated and cycloheximide-treated myotubes was carried out as described in the legend of Fig. 5, with Cy5-labeled primers located in MYH7b exons 5 and 9. Mobility of the PCR bands corresponding to splicing products that either include or skip exon 7 is indicated on the right. (D) Representative RT-PCR (33 cycles) from RNA isolated from untreated and cycloheximide-treated myotubes carried out to detect only transcripts including exon 7. For this experiment the Cy5-labeled forward primer was located in MYH7b exon 5 while the reverse primer was located at the junction of exons 7 and 8. The scheme of the splicing product detected by PCR that includes exon 7 is indicated on the right.

FIG. 8.

Analysis of miR-499 predicted targets. (A) COS-7 cells were cotransfected with the pGL-Sox6 reporter, containing 1 kb of the Sox6 3′ UTR fused to the firefly luciferase gene, and the empty vector pcDNA3 used as negative control (C) or an miR-499 overexpression construct (499). Firefly luciferase activity measured 24 h after transfection was normalized to Renilla luciferase activity. Reporter activity was significantly decreased after miR-499 overexpression compared to control (P < 0.01). Error bars indicate the standard errors of the means calculated from four independent experiments. (B) Neonatal rat cardiomyocytes were infected with a negative-control virus expressing β-galactosidase (β-gal) or a recombinant adenovirus expressing miR-499 (499). Sox6 real-time PCR was performed on total RNA purified 2 days after viral infection. Reactions were normalized to the 18S rRNA. Sox6 mRNA was significantly reduced after miR-499 overexpression (P < 0.05); error bars indicate the standard errors of the means calculated from three independent experiments. (C) C2C12 myoblasts were cotransfected with the MYH7b 1-kb promoter and the empty vector pcDNA3 used as negative control (C) or the expression vector pcSox6 (Sox6). MYH7b promoter activity was significantly decreased after Sox6 overexpression (P < 0.05); error bars indicate the standard errors of the means calculated from four independent experiments. (D) C2C12 myoblasts were transfected with the empty vector pcDNA3 (Ctrl) or the expression vector pcSox6 (Sox6). Levels of MYH7b, MYH-β, and MYH-IIb mRNA were determined 48 h postdifferentiation by real-time PCR. Each reaction was normalized to the 18S rRNA (relative units). Expression of all three genes significantly changed after Sox6 overexpression (P < 0.01); error bars indicate the standard errors of the means calculated from four independent experiments. (E) A representative miRNA Northern blot of total RNA extracted from C2C12 after transfection with the empty vector pcDNA3 (C) or pcSox6 (S) and 48 h of differentiation is shown. U6 snRNA was used as a loading control. (F) Quantification of the Northern blot analysis shown in panel E calculated from four independent experiments. Error bars indicate the standard errors of the means. (G) Validation of additional miR-499 predicted targets. COS-7 cells were cotransfected with a pGL3 reporter containing the 3′ UTR of Thrap1, myostatin (Mstn), Mapk6, and Sp3 with an empty expression plasmid control (C) or miR-499 expression construct (499). Reporter firefly luciferase activity was measured and normalized as above. Activity of all four reporters was significantly reduced after miR-499 overexpression (P < 0.05). Error bars indicate the standard errors of the means calculated from at least three independent experiments.