New tertiary constraints between the RNA components of active yeast spliceosomes: a photo-crosslinking study.
Elucidation of the three-dimensional (3D) structures of the two sequential active sites in spliceosomes is essential for understanding the mechanism of premessenger RNA splicing. The mechanism is predicted to be catalyzed by the small nuclear RNA (snRNA) components of spliceosomes. To obtain new tertiary constraints between the RNA components, we produced and mapped crosslinks between U6 snRNA and the proximal RNAs of active yeast spliceosomes ("yeast" in this report is Saccharomyces cerevisiae). Thus, specific sites in U6, when substituted with a photoreactive 4-thiouridine or 5-iodouridine, produced spliceosome-dependent crosslinks to U2 snRNA, or in one case, to the pre-mRNA substrate. One set of U2-U6 crosslinks formed before the Prp2p-dependent step of spliceosome assembly, whereas another set formed during or after this step but before the first chemical step of splicing. This latter set of crosslinks formed across U2-U6 helix I. Importantly, this set provides new tertiary constraints for developing 3D models of fully assembled yeast spliceosomes, which are poised for the first chemical step of splicing.
Ryan
Daniel E
DE
Division of Biology 147-75, California Institute of Technology, Pasedena 91125, USA. danieleryan@earthlink.net
Kim
Chang Hee
CH
Murray
James B
JB
Adams
Chris J
CJ
Stockley
Peter G
PG
Abelson
John
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D053487:DEAD-box RNA Helicases; D007065:Idoxuridine / Q000031:analogs & derivatives* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012326:RNA Splicing / Q000235:genetics* / Q000502:physiology; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017381:Spliceosomes / Q000235:genetics*
D012342:RNA, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA; C405486:U6 small nuclear RNA; C035991:5-iodouridine; D000255:Adenosine Triphosphate; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D007065:Idoxuridine