Fig 2: SPT6 helps to recruit Integrator to the human snRNA genes. (a) Re-analysis of previously published ChIP-seq of pol II (left) and SPT6 (right) over the U2 snRNA genes (RNU2) and other snRNA genes in 293 CDK12as cells [43]. (b) ChIP-qPCR of SPT6 across the U2 snRNA genes in 293 cells. The PSE value was normalised to 1. (c) Re-analysis of previously published ChIP-seq of INTS3 across the U2 snRNA genes and other snRNA genes in HeLa cells with and without siRNA-mediated knockdown of SPT6 [42]. (d) Re-analysis of previously published chromatin RNA-seq over the U2 snRNA genes and other snRNA genes in HeLa cells with and without siRNA-mediated knockdown of SPT6 [41].

Fig 3: CDK12 activity is required for stable association of elongation factors with chromatin. (A) Meta-analysis of LEO1 subunit of the PAF1 complex (PAF1C) ChIP-seq across scaled expressed genes. (B) Meta-analysis of SPT6 ChIP-seq across scaled expressed genes. (C) Meta-analyses of LEO1 ChIP-seq ratioed to the total pol II signal before and after CDK12as inhibition for 15 min across scaled expressed genes for the different length classes noted. (D) Meta-analyses of SPT6 ChIP-seq ratioed to the total pol II signal before and after CDK12as inhibition for 15 min across scaled expressed genes for the different length classes noted. (E) Western blots of CDK12as cell chromatin extracts. Cells are either treated with 7.5 μM 1-NM-PP1 or DMSO for 15 and 30 min as noted. The antibodies used are indicated on the right. Histone H3 was used as a loading control. (F) Quantitation of chromatin protein levels of SPT6, LEO1, CDC73, and histone H3 in CDK12as cells relative to DMSO controls. Error bars = s.e.m. (n = 3 biological replicates). Statistical test: two-tailed unpaired t test, ns = not significant, * P < 0.05, *** P < 0.001.

Fig 4: Model of CDK12 function in transcription. During the transcription cycle, CDK12 phosphorylates Ser2 and Ser5 of the pol II CTD and possibly other factors involved in transcription such as SPT6. Inhibition of CDK12 affects the pol II elongation rate by affecting the recruitment and/or stability on the chromatin of LEO1 and CDC73 from the PAF1 complex (PAF1C) and SPT6. CDK12 inhibition does not affect pol II pause release, which is mediated by CDK9. Orange line with ‘cap’: capped mRNA; orange and red dot: Ser2P and Ser5P, respectively. Red and violet arrows: phosphorylation mediated by CDK9 and CDK12, respectively.

Fig 5: MAGI2-AS3 induced antitumor effects in LSCC cells by interacting with SPT6. (A) Schematic of the MS2-12X-dependent RNA pull-down experiments. (B) MAGI2-AS3-binding proteins by mass spectrometry analysis. (C) SPT6 identified specifically bind to MAGI2-AS3 by Western blotting. (D) RIP was performed to confirm the interaction of SPT6 and MAGI2-AS3. (E) The expression levels of SPT6 in the TU177 knockdown of SPT6. (F, G) TU177 and AMC-HN-8 cells were transfected with oe-MAGI2-AS3 or cotransfected with oe-MAGI2-AS3 and sh-SPT6. Cell proliferation was obtained by the MTS assay. (H) Colony formation assays were performed to assess the proliferative ability of TU177 and AMC-HN-8 cells transfected with oe-MAGI2-AS3 or cotransfected with oe-MAGI2-AS3 and sh-SPT6. Effects of oe-MAGI2-AS3 and sh-SPT6 on the migration and invasion of TU177 and AMC-HN-8 cells were assessed by the (I, J) wound-healing assay and (K, L) transwell migration and invasion assays. Scale bar, 100 µm. Data are presented as mean ± SD of three independent experiments. *Vector versus oe-MAGI2-AS3; #oe-MAGI2-AS3 versus oe-MAGI2-AS3+sh-SPT6; *# Vector versus oe-MAGI2-AS3+sh-SPT6. *P < 0.05, **P < 0.01; #P < 0.05, ##P < 0.01; *#P < 0.01. MAGI2-AS3: MAGI2 antisense RNA 3; LSCC: laryngeal squamous cell carcinoma; SPT6: Suppressor Of Ty 6 Homolog; RIP: RNA Immunoprecipitation; AMC-HN-8: Asian Medical Center-Head and Neck cancer-8; GFP: green fluorescent protein.