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Abstract

Abstract After excising an intron from pre-mRNA, the spliceosome remains trapped in a non-productive complex bound to the intron 1–5 . Termination of this complex is critical for spliceosome recycling and intron decay 6 , but the mechanism remains unknown. Here we present cryo-electron microscopy structures of human spliceosomes at two sequential stages of termination. First, the RNA helicases DHX15 and Aquarius unwind the RNA active site of the spliceosome, releasing bound components including U2 snRNA and extracting the buried intron–lariat branch point. The branch point is then debranched by the spliceosome-tethered enzyme DBR1, generating the previously unknown debranched intron spliceosome. This state recruits the RNA helicase DHX35 with its co-factors GPATCH1–WDR83, assisted by YJU2B. DHX35 ejects the debranched intron from the U6 snRNA–5′ splice site duplex, driving spliceosome disassembly and intron turnover. In defective spliceosomes stalled on aberrant introns, YJU2B partners with LENG1 to guide DHX35–GPATCH1–WDR83 for termination through spliceosome quality control. Together, we reveal the mechanism of regular spliceosome termination and its parallels with spliceosome quality control, ensuring accurate and efficient pre-mRNA splicing.