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Specific mRNP complexes. Characterization of the proteins bound to histone H4 mRNAs isolated from L6 myoblasts
Abstract:
These studies were designed to identify the proteins associated with specific mRNAs. L6 myoblasts contain a unique poly(A)-rich H4 mRNA as well as poly(A)-minus H4 mRNA subspecies. We have characterized the proteins present in both poly(A)-rich and poly(A)-minus histone H4 mRNP complexes following ultraviolet cross-linking in vivo. In addition, the muscle-specific myosin heavy chain (MHC) mRNP complex was characterized in myoblasts. [35S]Methionine-labelled poly(A)-rich and poly(A)-minus RNP complexes were prepared from both the polysomal and free (post-polysomal) RNP compartments. From each fraction the mRNP encoding histone H4 or MHC was purified by hybrid selection to a cloned human histone H4 gene or MHC cDNA. A unique set of 6-16 proteins was found bound to each of the specific mRNP complexes. These proteins were a subset of the total population of either polysomal or free RNP proteins and some proteins appeared common among the different hybrid-selected RNP fractions. The results demonstrate that (a) mRNAs bind a different set of proteins depending upon whether they are present in the polysomal or free mRNP fraction; (b) the presence of poly(A) sequences affects the proteins which bind to H4 mRNA in the free RNP compartment.
Insights
Messenger ribonucleoprotein (mRNP) complexes bind distinct proteins based on mRNA location and polyadenylation status. This study identifies specific protein sets associated with histone H4 and myosin heavy chain (MHC) mRNAs in myoblasts.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Messenger RNAs (mRNAs) exist in cells as complexes with proteins, forming messenger ribonucleoprotein (mRNP) complexes.
- These mRNP complexes are involved in various post-transcriptional processes, including mRNA localization, translation, and stability.
- Specific mRNAs can exist in different forms, such as polyadenylated and non-polyadenylated versions, which may influence their associated proteins.
Purpose of the Study:
- To identify and characterize the specific proteins bound to distinct mRNA molecules within mRNP complexes.
- To investigate how the polyadenylation status of an mRNA affects its associated protein composition.
- To compare protein binding patterns between polysomal and free mRNP fractions.
Main Methods:
- Ultraviolet (UV) cross-linking in vivo to capture protein-mRNA interactions.
- Isolation and purification of specific mRNP complexes using hybrid selection with cloned genes (histone H4) and complementary DNA (cDNA) (myosin heavy chain, MHC).
- Analysis of radiolabeled ([35S]Methionine) proteins associated with purified mRNP complexes from polysomal and free RNP compartments.
Main Results:
- A unique set of 6-16 proteins was identified for each specific mRNP complex studied (histone H4 and MHC).
- The protein composition of mRNP complexes differs depending on whether they are in the polysomal or free RNP fraction.
- The presence or absence of poly(A) sequences influences the proteins that bind to histone H4 mRNA in the free RNP compartment.
Conclusions:
- mRNAs recruit different protein partners based on their subcellular localization (polysomal vs. free RNP).
- Polyadenylation status is a critical factor determining protein association with specific mRNAs, particularly in the free RNP fraction.
- These findings reveal a layer of regulatory complexity in gene expression mediated by mRNA-protein interactions.