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Article https://doi.org/10.1038/s41467-024-55063-7DSIF factor Spt5 coordinates transcription,maturation and exoribonucleolysis of RNApolymerase II transcriptsKrzysztof Ku?1, Loic Carrique2 , Tea Kecman 1 , Marjorie Fournier 1 ,Sarah Sayed Hassanein 1,3 , Ebru Aydin4 , Cornelia Kilchert 4 ,Jonathan M. Grimes2& Lidia Vasiljeva1Precursor messenger RNA (pre-mRNA) is processed into its functional formduring RNA polymerase II (Pol II) transcription. Although functional couplingbetweentranscriptionandpre-mRNAprocessingisestablished,theunderlyingmechanisms are not fully understood. We show that the key transcriptiontermination factor, RNA exonuclease Xrn2 engages with Pol II forming a stablecomplex. Xrn2 activity is stimulated by Spt5 to ensure eff i cient degradation ofnascent RNA leading to Pol II dislodgement from DNA. Our results support amodel where Xrn2 f i rst forms a stable complex with the elongating Pol II toachieveitsfullactivityindegradingnascentRNArevisingthecurrent‘torpedo’model of termination, which posits that RNA degradation precedes Xrn2engagementwith Pol II. Spt5 is also a key factor that attenuates the expressionof non-coding transcripts, coordinates pre-mRNA splicing and 3’-end proces-sing. Our f i ndings indicate that engagement with the transcribing Pol II is anessentialregulatorystepmodulatingtheactivityofRNAenzymessuchasXrn2,thusadvancingourunderstandingofhowRNAmaturationiscontrolledduringtranscription.DNA-dependent RNA polymerase II (Pol II) is responsible for thetranscription of protein-coding (mRNA) and non-coding RNA(ncRNA) in eukaryotic cells. ncRNAs represent a diverse class oftranscripts including stable house-keeping ncRNAs such as smallnuclear (sn)RNAs and unstable ncRNAs encompassing upstreamtranscriptsderivedfrombi-directionalpromoters 1 .BothclassesofPolII transcription units (TU) undergo processing to become functionalmolecules. Precursor messenger RNA (pre-mRNA) processing takesplace throughout the Pol II transcription cycle which consists ofinitiation, elongation, and termination stages. The coupling of pre-mRNA processing to transcription controls the timely recruitment ofRNAprocessingfactorsaswellasf i delity,eff i ciency,andregulationofthe RNA-processing reactions 2–6 . Defective RNA processing is linkedto neurodegenerative disorders and cancer 4,7–9 , however, themechanistic understanding of how Pol II transcription controls RNAprocessing is limited.Shortly after transcription initiation, pre-mRNA undergoes 5’-m 7 Gcapping, whereas splicing occurs during elongation. At the 3’-end ofgenes, pre-mRNA is cleaved and polyadenylated by the Cleavage andPolyAdenylation machinery (CPA). Recognition of the polyadenylationsignal (PAS) and pre-mRNA cleavage by CPA is linked to Pol II dislodge-ment and termination of transcription 10–13 . PAS consists of AAUAAA andGUelementsrecognisedbyCPAandCleavageFactorsAandB,CFAandBin yeast and CFI and CFII in mammals 12–15 . The endonuclease subunit oftheCPA(mammalianCPSF73andyeastYsh1)cleavesRNAbetweenthesetwo elements generating a 5’-monophosphorylated RNA end that iscrucial for initiating 5?–3? degradation of the Pol II-associated RNA byexoribonuclease Xrn2. It was proposed that Xrn2 ‘chases’ transcribingReceived: 15 November 2023Accepted: 29 November 2024Check for updates1 Department of Biochemistry, University of Oxford, Oxford, United Kingdom. 2 Division of Structural Biology, Wellcome Trust Centre for Human Genetics,University of Oxford, Oxford, United Kingdom.3 Zoology Department, Faculty of Science, Cairo University, Giza, Egypt. 4 Institut für Biochemie, Justus-Liebig-Universität Gießen, Gießen, Germany. e-mail: krzysztof.kus@bioch.ox.ac.uk; lidia.vasilieva@bioch.ox.ac.ukNature Communications| (2025) 16:10 11234567890():,;1234567890():,;