Binding of an RNA trafficking response element to heterogeneous nuclear ribonucleoproteins A1 and A2.
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K(d) near 50 nm, whereas the K(d) for hnRNP A1 was above 10 microm. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 microm for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.
Shan
J
J
Biochemistry Department, The University of Queensland, Queensland 4072, Australia.
Moran-Jones
K
K
Munro
T P
TP
Kidd
G J
GJ
Winzor
D J
DJ
Hoek
K S
KS
Smith
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006493:Heparin / Q000494:pharmacology; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D004676:Myelin Basic Protein; D009838:Oligodeoxyribonucleotides; D020033:Protein Isoforms; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C439768:hnRNP A2; D006493:Heparin