Oligonucleotide binding specificities of the hnRNP C protein tetramer

Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian 40S 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...

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Bibliographic Details
Published in:Nucleic acids research 1998-07, Vol.26 (14), p.3410-3417
Main Authors: Soltaninassab, Syrus R., McAfee, James G., Shahied-Milam, Lillian, LeStourgeon, Wallace M.
Format: Article
Language:English
Subjects:
Adenovirus
Base Sequence
Biopolymers
HeLa Cells
Heterogeneous-Nuclear Ribonucleoprotein Group C
Heterogeneous-Nuclear Ribonucleoproteins
Humans
Oligonucleotides - metabolism
Protein Binding
Ribonucleoproteins - chemistry
Ribonucleoproteins - metabolism
Spectrometry, Fluorescence
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Summary:Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian 40S 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 α-tropomyosin, the consensus polypyrimidine tract for U2AF65, AUUUA repeats and r(U)20 were used as competitors. A 20 nt β-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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/26.14.3410