The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.

译文

剪接体的小核RNA (snRNA) 成分在其组装,催化活化和拆卸过程中会经历许多构象重排。U4和U6 snrna作为U4/U6.U5小核核糖核蛋白 (tri-snRNP) 内的碱基配对复合物掺入剪接体中。然后将U4和U6展开,以便U6与U2配对以形成剪接体的活性位点。拼接后,将U2/U6展开,并将U6退火至U4以重新组装tri-snRNP。U6重排对于剪接体的形成至关重要,但知之甚少。我们已经使用单分子f ö rster共振能量转移和退绕测定法来鉴定促进U4/U6退绕的相互作用,并研究了它们在酵母中的影响。我们发现,通过将U4/U6茎II的退绕与茎I的链侵袭偶联,使用DNA寡核苷酸有效地解开了U4/U6。U6远干刺激解旋,该远干在完整的U4/U6 RNA复合物中瞬时形成。体内远端的稳定会导致U4/U6 di-snRNP的积累并损害酵母的生长。我们的数据揭示了U4/U6退绕的保守机制,并表明远程动力学对于tri-snRNP组装和稳定性至关重要。

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