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Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells
A Kumar1, H Sierakowska, W Szer
1Department of Biochemistry, New York University School of Medicine, New York 10016.
Insights
Type C protein, a minor heterogeneous nuclear ribonucleoprotein (hnRNP), binds RNA and prefers guanine (G)-rich and uracil (U)-rich sequences. This protein plays a role in pre-mRNA processing by interacting with specific nucleotide regions.
Area of Science:
- Molecular Biology
- Biochemistry
- RNA Processing
Background:
- Heterogeneous nuclear ribonucleoprotein (hnRNP) particles are crucial for RNA processing.
- Type C protein is a minor component of hnRNP particles.
Purpose of the Study:
- To purify and characterize a Type C protein from HeLa hnRNP particles.
- To investigate the RNA binding properties and nucleotide specificity of Type C protein.
Main Methods:
- Purification of Type C protein under nondenaturing conditions.
- RNA binding assays using natural and synthetic polynucleotides.
- Analysis of protein binding affinity for various nucleotide sequences.
Main Results:
- Type C protein was purified to near homogeneity.
- The protein disrupts RNA secondary structure and decreases the melting temperature (Tm) of RNA duplexes.
- Type C protein exhibits high affinity for G-rich and U-rich sequences, with a preference for G-rich regions.
- Binding is not highly cooperative, with a stoichiometry of one protein per approximately 10 nucleotides.
- Adenine (A) residues do not interfere with binding, but cytosine (C)-rich regions inhibit binding.
Conclusions:
- Type C protein's nucleotide specificity suggests a role in pre-mRNA processing, particularly at splice sites.
- The protein's preference for G/U-rich regions aligns with the composition of certain pre-mRNA processing signals.
Abstract:
A protein of the C group, most likely C3 (Mr approximately 42,000, pI approximately 6, corresponding to IEF 48m,n of the HeLa protein catalogue (Celis, J. E., Bravo, R., Arenstorf, H. P., and LeStourgeon, W. M. (1986) FEBS Lett. 194, 101-109)), a minor hnRNP protein was purified to near homogeneity under nondenaturing conditions from 40 S heterogeneous nuclear ribonucleoprotein particles. Type C protein stoichiometrically disrupts the residual secondary structure of natural and synthetic RNAs, e.g. HeLa hnRNA, coliphage MS2 RNA, and poly(rU)-spermine, and decreases the Tm of duplex structures, e.g. poly[r(A + U)], by about 30 degrees C. Binding of the protein to polynucleotides is not highly cooperative and has a stoichiometry of one protein per about 10 nucleotides. Binding experiments with a variety of synthetic and natural poly- and oligonucleotides, including those containing consensus splice site sequences, indicate that the protein has a high affinity for G-rich and U-rich regions, G-rich regions being preferred. Base analogs I and T have affinities for the protein that are similar to G and U. There is little or no affinity for A- and C-rich regions. The presence of A residues in a G- or U-rich sequence does not interfere with binding while C-rich regions decrease or prevent the binding of the protein. The nucleotide specificity of type C protein, e.g. selective binding to an oligonucleotide from the 3' end of an intron, is discussed in relationship to the abundance of G and U and the relative scarcity of C residues in the processing signals in pre-mRNA.