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Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3′-end processing factors for RDH genes are enriched; however, this association’s role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs’ association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)–Cap binding complex (CBC)–Negative elongation factor (NELF), components of CBs, interferes with CBs’ association with HLBs and 3′ Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator’s involvement in CBs’ association with HLBs to facilitate 3′ Pol II pausing and subsequent 3′-end processing of RDH genes by supplying 3′-end processing factors.

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