SRP485654 PRJNA1068524 GSE254080 TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing [CUT&RUN] TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that the TAF2 IDR specifically interacts with the nuclear speckle and spliceosome-associated protein SRRM2. Consequently, TAF2 recruits SRRM2 to TFIID to form non-canonical TFIID-SRRM2 complexes. Reduced SRRM2 recruitment elicits alternative splicing events in RNAs coding for proteins involved in transcription and transmembrane transport. Further, genome-wide binding analyses suggest TAF2 shuttling between nuclear speckles and pol II promoters. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing. Overall design: Cleavage under targets and release using nuclease (CUT&RUN) and greenCUT&RUN followed by DNA sequencing for TFIID subunits TAF1, TAF2, TAF7, TBP and RNA polymerase II subunit RPB1 (CUT&RUN) and for GFP-TAF2, GFP-NLS-TAF2, GFP-NLS-TAF2delta1009-1199 (2 replicates), GFP-NLS-TAF2delta1142-1171 (2 replicates) (greenCUT&RUN). Each CUT&RUN sample has a respective IgG control sample. Each greenCUT&RUN sample has a respective control sample (-Ca2+). All libraries contain spike-in DNA from Drosophila melanogaster. HeLa Flp-In T-Rex cells harboring a transgene under the control of a doxycycline-inducible promoter were used. Protein expression was induced with 1 ug/mL doxycycline where appropriate. GSE254080 parent_bioproject PRJNA1068517