Oligonucleotide binding specificities of the hnRNP C protein tetramer.
Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian40S hnRNP particles has been characterized previously as a poly(U) binding protein with specificity for the pyrimidine-rich sequences that often precede 3' intron-exon junctions. C protein has also been characterized as a sequence-independent RNA chaperonin that is distributed along nascent transcripts through cooperative binding and as a protein ruler that defines the length of RNA packaged in 40S monoparticles. In this study fluorescence spectroscopy was used to monitor C protein-oligonucleotide binding in a competition binding assay under equilibrium conditions. Twenty nucleotide substrates corresponding to polypyrimidine tracts from IVS1 of the adenovirus-2 major late transcript, the adenovirus-2 oncoprotein E1A 3' splice site, IVS2 of human alpha-tropomyosin, the consensus polypyrimidine tract for U2AF65, AUUUA repeats and r(U)20were used as competitors. A 20 nt beta-globin intronic sequence and a randomly generated oligo were used as competitor controls. These studies reveal that native C protein possesses no enhanced affinity for uridine-rich oligonucleotides, but they confirm the enhanced affinity of C protein for an oligonucleotide identified as a high affinity substrate through selection and amplification. Evidence that the affinity of C protein for the winner sequence is due primarily to its unique structure or to a unique context is seen in its retained substrate affinity when contiguous uridines are replaced with contiguous guanosines.
Soltaninassab
S R
SR
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA.
McAfee
J G
JG
Shahied-Milam
L
L
LeStourgeon
W M
WM
D001483:Base Sequence; D001704:Biopolymers; D006367:HeLa Cells; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D009841:Oligonucleotides / Q000378:metabolism*; D011485:Protein Binding; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001704:Biopolymers; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009841:Oligonucleotides; D012261:Ribonucleoproteins