hnRNP A1 binds promiscuously to oligoribonucleotides: Utilization of random and homo-oligonucleotides to discriminate sequence from base-specific binding
hnRNP A1 binds promiscuously to oligoribonucleotides: Utilization of random and homo-oligonucleotides to discriminate sequence from base-specific binding
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DOI：
10.1093/nar/24.20.4063
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发表时间：
1996-10-15
影响因子：
14.9
通讯作者：
Williams, KR
中科院分区：
文献类型：
--
作者：
AbdulManan, N;Williams, KR
为了了解前体mRNA生物发生中可能的和可能的Al功能的范围，重要的是我们量化了前体mRNA生物发生中Al功能的相对能力。Al结合高亲和力RNA靶序列和/或结构的能力（或无能力），使用荧光竞争测定法，我们已经确定了含有推定的和可能的Al靶的广泛的20聚体寡核苷酸的表观结合亲和力，所述靶包括由以下鉴定的高亲和力“赢家”序列：选择/扩增[Burd，C，G和Dreyfuss，G.（1994）EMBO J. 13，1197-1204]，在不稳定mRNA的3 '-UTR中发现的AUUUA序列、5'-和3 '-剪接位点和端粒序列。除了20聚体“赢家”序列之外，所有其他检查的20聚体以窄的，- 从3.2 X 10（6）延伸至4.2 X 10（7）M（-1）的10倍亲和力范围。同源寡聚体的研究表明，该范围反映了核苷酸碱基而不是序列特异性，因此，可以合理准确地预测所有其他20聚体的亲和力，但“获胜者”除外，其4.0 x 10（8）M（-1）的异常高亲和力来自独特的高级结构和序列，由于“赢家”20聚体序列没有已知的生理作用，这些数据表明Al通常不加区别地结合所有可用的前mRNA序列，因此，体内大量的Al及其结合特性与其在前mRNA生物发生中发挥结构作用一致。
To understand the range of possible and probable Al functions in pre-mRNA biogenesis, it is important that we quantify the relative ability (or inability) of Al to bind high affinity RNA target sequences and/or structures, Using a fluorescence competition assay we have determined apparent binding affinities for a wide range of 20mer oligos containing putative and possible Al targets including the high affinity 'winner' sequence identified by selection/amplification [Burd,C,G and Dreyfuss,G. (1994) EMBO J. 13, 1197-1204], AUUUA sequences found in 3'-UTRs of labile mRNAs, 5'- and 3'-splice sites and telomeric sequences, With the exception of a 20mer 'winner' sequence, all other 20mers examined bind Al with a narrow, -10-fold range of affinities extending from 3.2 x 10(6) to 4.2 x 10(7) M(-1). Studies with homo-oligomers suggest this range reflects nucleotide base rather than sequence specificity and hence, it was possible to predict reasonably accurate affinities for all other 20mers examined except for the 'winner', whose unusually high affinity of 4.0 x 10(8) M(-1) results from a unique higher order structure and sequence, Since there is no known physiological role for the 'winner' 20mer sequence, these data suggest Al generally binds indiscriminately to all available pre-mRNA sequences, Both the large abundance of Al in vivo and its binding properties are thus consistent with it playing a structural role in pre-mRNA biogenesis.