期刊
EMBO JOURNAL
卷 25, 期 11, 页码 2475-2486出版社
WILEY
DOI: 10.1038/sj.emboj.7601134
关键词
pre-mRNA splicing; site-directed hydroxyl-radical probing; spliceosome
Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA-RNA interactions, little is known about the tertiary structure of this RNA-RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide ( U+10) of the 50 end of a pre-mRNA intron, to map RNA-RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U+10 in activated spliceosomes, namely ( i) the U6 snRNA ACAGAG-box region, ( ii) portions of the U6 intramolecular stem-loop ( U6-ISL) including a nucleotide implicated in the first catalytic step ( U74), and ( iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U+10 in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step.
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