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Quantitation of muscle-specific mRNAs by using cDNA probes during chicken embryonic muscle development in ovo
Abstract:
The emergence of abundant-class mRNAs specific for contractile muscle proteins and their distribution between polysomal and free mRNP fractions were studied in skeletal muscle excised from chicken embryos during the transition from myoblasts (day 9) to myotubes (day 18). Muscle-specific cDNA was selectively prepared by hybridizing cDNA to template RNA (polysomal poly(A)+ mRNA) from day-14 embryos followed by isolation of the abundant class, which represents approximately 20% of total mRNA. The specificity of the cDNA probe for this class was confirmed by the differential degree of hybridization to cytoplasmic RNA from cultured myotube and myoblast cells and by its inability to hybridize with mRNA from nonmuscle cells such as liver. Except for muscle from day-9 embryos, the concentrations of the abundant-class muscle-specific mRNAs were higher in polysomes than in free mRNP fractions. Furthermore, the levels of these mRNAs in polysomes increased 12-fold from day 9 (myoblast) to day 14 (intermediate) with a further 3.6-fold increase from day 14 to day 18 (myotube). In contrast to this 45-fold net increase in the polysomal level of these mRNAs from day 9 to day 18, the levels in the free mRNP fraction showed only a 3-fold decrease during this period. Because the amount of mRNA lost from the mRNP fraction is much less than the net increase in the polysome fraction, mRNP does not serve as a reservoir of untranslated muscle-specific mRNA for transfer to polysomes. Consequently, the emergence of muscle-specific polysomal mRNA for contractile proteins during myogenesis in ovo appears to be regulated primarily by transcriptional control.
Insights
During muscle development, abundant mRNAs for contractile proteins are primarily regulated by gene transcription, not stored in free messenger ribonucleoprotein (mRNP) particles. This ensures efficient protein synthesis for myogenesis.
Area of Science:
- Molecular Biology
- Developmental Biology
- Cellular Biology
Background:
- Skeletal muscle development involves the coordinated expression of contractile protein genes.
- Messenger RNA (mRNA) localization and translation are critical for protein synthesis during myogenesis.
- The role of free messenger ribonucleoprotein (mRNP) particles as a reservoir for untranslated mRNA during muscle differentiation is not fully understood.
Purpose of the Study:
- To investigate the emergence and distribution of abundant-class mRNAs specific for contractile muscle proteins during chicken embryo skeletal muscle development.
- To determine the relative contribution of transcriptional control versus post-transcriptional mechanisms (e.g., mRNA storage in mRNP) in regulating muscle-specific mRNA levels.
- To analyze the dynamic changes in polysomal and free mRNP fractions of these mRNAs from myoblast to myotube stages.
Main Methods:
- Preparation of muscle-specific complementary DNA (cDNA) probes by hybridizing cDNA to polysomal poly(A)+ mRNA from day-14 chicken embryos.
- Isolation of the abundant mRNA class, representing approximately 20% of total mRNA.
- Confirmation of cDNA probe specificity using differential hybridization to cytoplasmic RNA from cultured myotubes, myoblasts, and non-muscle liver cells.
- Quantification of abundant-class muscle-specific mRNA levels in polysomal and free mRNP fractions from skeletal muscle at different embryonic days (9, 14, and 18).
Main Results:
- Muscle-specific cDNA probes effectively identified abundant-class mRNAs for contractile proteins.
- Except in early myoblasts (day 9), muscle-specific mRNAs were predominantly found in polysomes rather than free mRNP fractions.
- Polysomal mRNA levels for contractile proteins showed a significant increase (45-fold) from day 9 to day 18.
- Free mRNP fractions exhibited only a minor decrease (3-fold) in these mRNAs during the same period.
- The increase in polysomal mRNA significantly exceeded the decrease in free mRNP-bound mRNA.
Conclusions:
- Free mRNP fractions do not serve as a significant reservoir for untranslated muscle-specific mRNA during myogenesis in ovo.
- The substantial increase in muscle-specific polysomal mRNA during myogenesis is primarily driven by transcriptional regulation.
- Gene transcription is the main regulatory mechanism controlling the availability of contractile protein mRNAs for translation during skeletal muscle differentiation.