Distinct regions in the 3' untranslated region are responsible for targeting and stabilizing utrophin transcripts in skeletal muscle cells

Abstract

In this study, we have sought to determine whether utrophin transcripts are targeted to a distinct subcellular compartment in skeletal muscle cells, and have examined the role of the 3' untranslated region (UTR) in regulating the stability and localization of utrophin transcripts. Our results show that utrophin transcripts associate preferentially with cytoskeleton-bound polysomes via actin microfilaments. Because this association is not evident in myoblasts, our findings also indicate that the localization of utrophin transcripts with cytoskeleton-bound polysomes is under developmental influences. Transfection of LacZ reporter constructs containing the utrophin 3'UTR showed that this region is critical for targeting chimeric mRNAs to cytoskeleton-bound polysomes and controlling transcript stability. Deletion studies resulted in the identification of distinct regions within the 3'UTR responsible for targeting and stabilizing utrophin mRNAs. Together, these results illustrate the contribution of posttranscriptional events in the regulation of utrophin in skeletal muscle. Accordingly, these findings provide novel targets, in addition to transcriptional events, for which pharmacological interventions may be envisaged to ultimately increase the endogenous levels of utrophin in skeletal muscle fibers from Duchenne muscular dystrophy (DMD) patients.

Figure 1.

Complete sequence of the utrophin 3′UTR. (A) Sequence of the mouse utrophin 3′UTR. (B) Comparison between the mouse 3′ UTR and the available rat and human sequences. The percentage of identity with the mouse sequence is also shown.

Figure 2.

Characterization of the polysomal fractions. Markers of the three distinct pools of polysomes were used to ensure the validity of the subcellular fractionation procedure. Activity of the cytosolic enzyme LDH was determined for the free polysomal fraction (text and Table I). (A) shows the levels of utrophin in the free (F), cytoskeletal (C)-, and membrane (M)-bound polysomal fractions as determined by Western blot analysis. (B) shows a representative ethidium bromide–stained agarose gel of RT-PCR products corresponding to α-AChR mRNAs obtained from the three fractions. The left lane is a 100-bp molecular mass marker. (−), negative control lane. Note the accumulation of utrophin in the cytoskeletal-bound fraction (B) and the enrichment of transcripts encoding the α-subunit of AChR in the membrane-bound polysomal fraction (C).

Figure 3.

Utrophin mRNAs preferentially associate with cytoskeletal-bound polysomes. (A) shows a representative ethidium bromide–stained agarose gel of RT-PCR products corresponding to utrophin mRNAs obtained from free (F), cytoskeletal (C)-, and membrane (M)-bound polysomal fractions. The left lane is a 100-bp molecular mass marker. (−), negative control lane. Note the accumulation of utrophin transcripts in the cytoskeletal-bound fraction. (B) Utrophin transcript levels in each fraction were determined and expressed as a percent of the total. Shown are the results of a minimum of six independent experiments.

Figure 4.

Utrophin mRNAs associate with actin microfilaments. (A and B) Representative examples of phalloidin-stained myotubes without and following exposure to Cy-D, respectively. Note the absence of fluorescence following Cy-D treatment. (C) Representative ethidium bromide-stained agarose gels of utrophin RT-PCR products obtained from RNA isolated from free (F), cytoskeletal (C)-, and membrane (M)-bound polysomes, along with a negative control (−). Left panel corresponds to polysomal fractions isolated from control (CTL) myotubes, revealing that utrophin mRNAs preferentially associate with the cytoskeleton. Right panel represents myotubes treated with CY-D showing a redistribution of utrophin mRNAs into the free polysomal fraction. (−), negative control lane. The left lane is a 100-bp molecular mass marker. Shown are representative results of a minimum of five independent experiments. Bar, 230 μm.

Figure 5.

Utrophin mRNAs do not preferentially associate with cytoskeletal-bound polysomes in myoblasts. (A) Representative ethidium bromide–stained agarose gels of PCR products corresponding to utrophin cDNAs obtained from free (F), cytoskeletal-bound (C), and membrane (M)-bound polysomal fractions. The left lane is a 100-bp molecular mass marker. (−), negative control lane. Note the lack of preferential accumulation of utrophin transcripts within the cytoskeletal-bound fraction. (B) Utrophin transcript levels in each polysomal fraction were determined and expressed as a percent of the total. Shown are the results of a minimum of seven independent experiments. (C) Utrophin transcript levels in polysomal fractions were also determined in myotubes treated with agrin and expressed as a percent of the total. Shown are the results of a minimum of four independent experiments.

Figure 7.

Targeting domain within the utrophin 3′UTR. (A) Representative examples of ethidium bromide–stained agarose gels corresponding to β-galactosidase RT-PCR products obtained from free (F), cytoskeletal (C)-, and membrane (M)-bound polysomal fractions after transfections with reporter constructs containing truncated fragments of the 3′UTR. Note that β-galactosidase constructs containing nucleotide sequence 332–596 are still targeted to cytoskeletal-bound polysomes and that this localization is lost with a smaller 3′UTR fragment (332 nt). (B) The levels of β-galactosidase transcripts for each polysomal fraction were determined and expressed as percentage of the total β-galactosidase mRNA levels detected. Shown are representative examples of a minimum of five experiments. (C) shows the reporter constructs used in these studies. The utrophin 3′UTR sequences were inserted downstream of the LacZ gene. Nucleotide positions are denoted on the right.

Figure 6.

The 3′UTR of utrophin mRNA targets LacZ transcripts to cytoskeletal-bound polysomes. (A) shows representative examples of ethidium bromide–stained agarose gels of β-galactosidase PCR products obtained from free (F), cytoskeletal (C)-, and membrane (M)-bound polysomes following transfections with constructs containing the utrophin 3′UTR in the forward or reverse orientation. (Left) Muscle cultures were transfected with the reporter construct containing the utrophin 3′UTR in the forward orientation. Note that in these myotubes, chimeric transcripts preferentially associate with cytoskeletal-bound polysomes. (Middle) Cultures were treated with Cy-D which disrupts this association as seen with endogenous utrophin mRNAs. (Right) β-galactosidase constructs containing the utrophin 3′UTR in the reverse orientation are not targeted to cytoskeletal-bound polysomes. (B) The levels of β-galactosidase transcripts for each polysomal fraction were determined and expressed as a percentage of the total β-galactosidase mRNA levels detected. Shown are the results obtained from a minimum of five independent experiments.

Figure 8.

The utrophin 3′UTR regulates the stability of β-galactosidase transcripts. β-Galactosidase reporter constructs containing the full-length utrophin 3′UTR were transfected into muscle cells in culture. The stability of β-galactosidase transcripts was determined by exposing cultures to actinomycin D at time zero. Samples were collected at various time points and total RNA was subsequently extracted and subjected to RT-PCR. A shows a representative ethidium bromide-stained agarose gel corresponding to β-galactosidase RT-PCR products. (−), negative lane. (B) Quantitation of these results revealed that the half-life of β-galactosidase transcripts containing the full-length utrophin 3′UTR is ∼22 h (○; line 1). Similar results were obtained with constructs containing the first 332 nt of the utrophin 3′UTR (lines 2, 4, and 5). Note also that the stability of the chimeric mRNAs was reduced with smaller fragments (lines 6 and 7) or when the region from nt 1 to 969 was absent from the 3′UTR (line 3). Shown are the pooled results from four independent experiments.