Purification and RNA binding properties of a C-type hnRNP protein from HeLa cells.
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.
D002942:Circular Dichroism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015245:Deoxyribonuclease BamHI; D006367:HeLa Cells / Q000032:analysis*; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007525:Isoelectric Focusing; D009690:Nucleic Acid Conformation; D010449:Peptide Mapping; D011072:Poly U / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012332:RNA, Heterogeneous Nuclear / Q000302:isolation & purification* / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000032:analysis
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D012261:Ribonucleoproteins; D011072:Poly U; D012313:RNA; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D006163:Ribonuclease T1