Investigation of the nonsense-mediated mRNA decay in the KI and Het mice

The experiments described in chapter 3.2.2 failed to provide evidence that the level of cMyBP-C mutants is regulated at the protein level in the KI mice. UPS inhibition did neither reveal the truncated protein nor increased the amount of the full-length mutant protein. On the other hand, the 78% decrease in the amount of total cMyBP-C mRNA in the KI mice (3.1.2.3) suggested that a strong regulation for the cMyBP-C mutants existed at the mRNA level. Thus, the nonsense-mediated mRNA decay (NMD; 1.5.1), which is assumed to play a key role in cell quality control by removal of PTC-containing nonsense mRNAs, was investigated in the KI and also in the Het mice, in which the nonsense mRNA was randomly detected. NMD occurs when the PTC is located more than 50-55 nt upstream of the last exon-exon junction (Nagy and Maquat, 1998). In KI and Het mice, the PTC is located in exon 9 within the nonsense mRNA and is therefore more than 55 nt upstream of the last junction between exons 34 and 35. Because NMD requires a pioneer round of translation in mammalian cells, it can be prevented by translation inhibitors like cycloheximide (CHX) or emetine (see 1.5.1). Therefore, cardiomyocytes were isolated from neonatal WT and KI mice, cultured and treated with CHX (100 µg/ml; Fig. 3.50) or emetine (300 µg/ml;

Fig. 3.51) for 4 h. After treatment, total RNA was isolated and analyzed by classical and quantitative RT-PCR. The level of total cMyBP-C transcripts was determined by quantitative RT-PCR using the SYBR^® Green strategy and primers located in exons 2 and 3 of the cMyBP-C cDNA. The level of nonsense mRNA was analyzed by quantitative RT-PCR using primers located in exons 5 and 7 of the cMyBP-C cDNA and a TaqMan^® probe, which matches the junction between exons 5 an 7 and is therefore specific for the nonsense mRNA. The level of missense mRNA was determined by quantitative RT-PCR using primers located in exons 6 and 7 of the cMyBP-C cDNA and a specific TaqMan^® probe located at the junction between exons 6 and 7 matching the G>A transition.

The basal level of total cMyBP-C transcripts was ~ 80% lower in KI than in WT cardiomyocytes (Fig. 3.50 A and B), which was comparable to the data obtained in adult hearts (Fig. 3.13). CHX treatment did not significantly affect the level of total cMyBP-C mRNA in both WT and KI cardiomyocytes (Fig. 3.50 A and B), but

Fig. 3.50: Effect of NMD inhibition by CHX on the mRNA level in cardiomyocytes of neonatal WT and KI mice. Cardiomyocytes were isolated from neonatal WT and KI mice, cultured and treated with CHX (100 µg/ml) or 0.1% DMSO for 4 h. A, The different cMyBP-C mRNA species were determined by classical RT-PCR using primers (black arrows) as indicated in the scheme on the right (miss means missense mRNA and nons nonsense mRNA). MW stands for the 100-bp molecular weight marker. B, The level of total cMyBP-C mRNA was determined by quantitative RT-PCR using the SYBR^® Green strategy and primers located in exons 2 and 3. C, The level of nonsense mRNA was determined by quantitative RT-PCR using primers (black arrows) and probe as indicated in the scheme below. Bars represent the mean±SEM with **P<0.01 vs. DMSO-treated cells, Student`s t-test. The number of preparations is indicated in the bars. These analyses were performed by Nicolas Vignier.

A

DMSO CHX

WT KI

DMSO CHX

363 bp 245 bp

WT 4 5 6 7 8 9

Miss

PTC 9 5 7 8 Nons 4

9 5 6 7 8 4

G>A MW

0.0 0.5 1.0 1.5

4 3 4 3

Total cMyBP-C mRNAlevel(AU)

DMSO CHX DMSO CHX

WT KI

B

0.0 0.5 1.0 1.5 2.0 2.5 3.0

5 4

**

NonsensemRNAlevel(AU)

DMSO CHX

KI C

PTC 9 5 7 8 Nons 4

A

DMSO CHX

WT KI

DMSO CHX

363 bp 245 bp

WT 44 55 66 7 87 8 99

Miss

PTC 9 5 7 8 Nons 4

PTC 9 5 7 8 4 5 7 8 9 Nons 4

9 5 6 7 8 4

G>A 9 5 6 7 8 4

G>A MW

0.0 0.5 1.0 1.5

4 3 4 3

Total cMyBP-C mRNAlevel(AU)

DMSO CHX DMSO CHX

WT KI

0.0 0.5 1.0 1.5

4 3 4 3

Total cMyBP-C mRNAlevel(AU)

DMSO CHX DMSO CHX

WT KI

B

0.0 0.5 1.0 1.5 2.0 2.5 3.0

5 4

**

NonsensemRNAlevel(AU)

DMSO CHX

KI 0.0

0.5 1.0 1.5 2.0 2.5 3.0

5 4

**

NonsensemRNAlevel(AU)

DMSO CHX

KI C

PTC 9 5 7 8 Nons 4

Fig. 3.51: Effect of NMD inhibition by emetine on the mRNA level in cardiomyocytes of neonatal WT and KI mice. Cardiomyocytes were isolated from neonatal WT and KI mice, cultured and treated with emetine (300 µg/ml; emet) or 0.1% DMSO for 4 h. A, The different cMyBP-C mRNA species were determined by classical RT-PCR using primers (black arrows) as indicated in the scheme (miss means missense and nons nonsense mRNA). MW stands for the 100-bp molecular weight marker. B, The level of total cMyBP-C mRNA was determined by quantitative RT-PCR using the SYBR^® Green strategy and primers located in exons 2 and 3. C, The level of missense mRNA was determined by quantitative RT-PCR using primers (black arrows) and probe as indicated in the scheme. D, The level of nonsense mRNA was determined by quantitative RT-PCR using primers (black arrows) and probe as indicated in the scheme. On the left, amplification curves of nonsense and GAPDH mRNA are shown. On the right, the statistical analysis is presented. Bars represent the mean±SEM with B

DMSO

WT KI

A

Emet DMSO Emet MW

363 bp 245 bp

WT 4 5 6 7 8 9

Miss

PTC 9 5 7 8 Nons 4

9 5 6 7 8 4

G>A

0.00 0.25 0.50 0.75 1.00 1.25

3 4 3

***

4

Total cMyBP-C mRNAlevel(AU)

WT KI

DMSO Emet DMSO Emet 0.0

0.5 1.0 1.5 2.0

3 4

MissensemRNAlevel(AU)

C

DMSO Emet

Miss 4 5 6 7 8 9 G>A D

Emet Ct 29.8

DMSO Ct 32.7

Amplification curve for nonsense mRNA

Amplification curve for GAPDH mRNA

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

3 4

***

NonsensemRNAlevel(AU)

PTC 9 5 7 8 Nons 4

DMSO Emet B

DMSO

WT KI

A

Emet DMSO Emet MW

363 bp 245 bp

WT 4 5 6 7 8 9

Miss

PTC 9 5 7 8 Nons 4

9 5 6 7 8 4

G>A WT 44 55 66 7 87 8 99

Miss

PTC 9 5 7 8 Nons 4

PTC 9 5 7 8 4 5 7 8 9 Nons 4

9 5 6 7 8 4

G>A 9 5 6 7 8 4

G>A

0.00 0.25 0.50 0.75 1.00 1.25

3 4 3

***

4

Total cMyBP-C mRNAlevel(AU)

WT KI

DMSO Emet DMSO Emet 0.0

0.5 1.0 1.5 2.0

3 4

MissensemRNAlevel(AU)

C

DMSO Emet

Miss 4 5 6 7 8 9 G>A D

Emet Ct 29.8

DMSO Ct 32.7

Amplification curve for nonsense mRNA

Amplification curve for GAPDH mRNA Emet

Ct 29.8

DMSO Ct 32.7

Amplification curve for nonsense mRNA

Amplification curve for GAPDH mRNA

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

3 4

***

NonsensemRNAlevel(AU)

PTC 9 5 7 8 Nons 4

DMSO Emet

Emetine did not change the level of total cMyBP-C mRNA in WT cells, but increased the level of total cMyBP-C transcripts 4-fold in KI cells (Fig. 3.51 A and B).

Quantitative analysis of the nonsense mRNA level in KI cells revealed a 7-fold increase after treatment with emetine as compared to DMSO (Fig. 3.51 D), whereas the missense mRNA level was not significantly changed after treatment (Fig. 3.51 C).

Both CHX and emetine treatment seemed to decrease the missense mRNA level as determined by the classical RT-PCR (Fig. 3.50 A and Fig. 3.51 A). However, this effect was likely due to a competition in the PCR reaction, because the specific TaqMan^® assay showed that emetine rather increased the missense mRNA level than decreased it (Fig. 3.51 C).

To analyze whether NMD is also involved in the degradation of the nonsense mRNA in vivo, CHX (120 mg/kg) or DMSO were 4 times (once per hour) subcutaneously injected in 3 WT, Het and KI mice. The animals were sacrificed 30 min after the last injection. Total RNA was isolated from the ventricles and analyzed by qualitative and quantitative RT-PCR (Fig. 3.52).

Fig. 3.52: Effect of NMD inhibition on the mRNA level in ventricles of CHX-treated WT, Het and KI mice. WT, Het and KI mice were subcutaneously injected with CHX (120 mg/kg, 4 times, once a hour) or 60% DMSO-NaCl-solution. Total ventricular RNA was then isolated. A, The different cMyBP-C mRNA species were determined by nested classical RT-PCR using primers (black arrows) as indicated in the scheme on the right (miss means missense mRNA and nons nonsense mRNA). MW stands for the 100-bp molecular weight marker. B, The level of total cMyBP-C mRNA was determined by quantitative RT-PCR using the SYBR^® Green strategy and primers located in exons 2 and 3. C, The level of missense mRNA was determined by quantitative RT-PCR using primers (black arrows) and probe as indicated in the scheme below. D, The level of nonsense mRNA was determined by quantitative RT-PCR using primers (black arrows) and probe as indicated in the scheme below. Bars represent the mean±SEM with *P<0.05 vs. DMSO-treated mice and ^###P<0.001 vs. DMSO-treated Het

DMSO CHX DMSO CHX DMSO CHX

WT Het KI

0.00 0.25 0.50 0.75 1.00 1.25

3 3 3 3 3 3

Total cMyBP-CmRNAlevel(AU)

DMSO CHX DMSO CHX DMSO CHX

WT Het KI

A

B

C D

363 bp 245 bp

WT

Miss

Nons

9

5 6 7 8

4

PTC 9 5 7 8 4

9

5 6 7 8

4

G>A MW

0 5 10 15 20 25 30 35

3 3 3 3

*

*

###

NonsensemRNAlevel(AU)

DMSO CHX Het

DMSO CHX KI

PTC 9 5 7 8 Nons 4

0.0 0.5 1.0 1.5

MissensemRNAlevel(AU)

3 3 3 3

DMSO CHX Het

DMSO CHX KI

Miss 4 5 6 7 8 9 G>A

DMSO CHX DMSO CHX DMSO CHX

WT Het KI

0.00 0.25 0.50 0.75 1.00 1.25

3 3 3 3 3 3

Total cMyBP-CmRNAlevel(AU)

DMSO CHX DMSO CHX DMSO CHX

WT Het KI

0.00 0.25 0.50 0.75 1.00 1.25

3 3 3 3 3 3

Total cMyBP-CmRNAlevel(AU)

DMSO CHX DMSO CHX DMSO CHX

WT Het KI

A

B

C D

363 bp 245 bp

WT

Miss

Nons

9

5 6 7 8

4

PTC 9 5 7 8 4

9

5 6 7 8

4

G>A WT

Miss

Nons

9

5 6 7 8

4

PTC 9 5 7 8 4

9

5 6 7 8

4

G>A 9

5 6 7 8

4

PTC 9 5 7 8 4

9

5 6 7 8

4

G>A MW

0 5 10 15 20 25 30 35

3 3 3 3

*

*

###

NonsensemRNAlevel(AU)

DMSO CHX Het

DMSO CHX KI 0

5 10 15 20 25 30 35

3 3 3 3

*

*

###

NonsensemRNAlevel(AU)

DMSO CHX Het

DMSO CHX KI

PTC 9 5 7 8 Nons 4

0.0 0.5 1.0 1.5

MissensemRNAlevel(AU)

3 3 3 3

DMSO CHX Het

DMSO CHX KI 0.0

0.5 1.0 1.5

MissensemRNAlevel(AU)

3 3 3 3

DMSO CHX Het

DMSO CHX KI

Miss 4 5 6 7 8 9 G>A

In vivo treatment with CHX did not significantly affect the level of total cMyBP-C transcripts in the WT and Het mice, but induced a tendency towards an increase (P=0.09) in the KI mice (Fig. 3.52 A and B). Quantitative analysis of the nonsense mRNA level revealed a 4-fold increase in the KI and a 6-fold increase in the Het mice after treatment with CHX as compared to the DMSO-treated mice (Fig. 3.52 D). Like in cells, the level of the missense mRNA was not changed by CHX in the KI (Fig.

3.52 C). Surprisingly, the missense mRNA was not detectable in the Het mice by TaqMan^® analysis, which would suggest that its level was very low compared to the WT mRNA (Fig. 3.52 C).

Im Dokument Investigation of molecular mechanisms regulating the expression of cMyBP-C mutants in familial hypertrophic cardiomyopathy (Seite 111-116)