Fig 2: A cluster of conserved residues is critical for Ints6 binding.A, Cartoon and (B) surface representations showing the distribution of conserved residues from ConSurf analysis (33). Each molecule contributes a stretch of conserved residues (dotted oval), and two such stretches are close together forming a larger patch. C, on the opposite side of this conserved patch is the basic groove proposed to bind ssRNA, which is moderately conserved. D, enlarged view of the conserved surface from molecule A. Positions of the conserved residues and mutations used in this study are indicated. Of these, R863/D869, later found to be dispensable for Ints6 binding, is slightly separated from the other more congregated ones. E, pull-down of Ints6-807-887 by GST-tagged Ints3 C-terminal domain mutants (m1-m5). GST-tagged Ints3 was first immobilized on glutathione Sepharose beads and then incubated with a purified MBP-tagged Ints6-807-887 protein. After extensive washing, MBP-Ints6-807-887 bound to Ints3 was detected using an MBP antibody. Representative result from at least three repeats is shown. F, Coimmunoprecipitation between full-length Flag-tagged wild-type or mutant Ints3 and V5-tagged Ints6. Immunoprecipitations (IPs) were performed with anti-Flag M2-agarose beads, and precipitated proteins were detected with a V5 antibody. Representative result from at least three repeats is shown. G, similar as in (F), but IPs were performed with anti-V5 agarose beads, and precipitated proteins were detected with a Flag antibody.

Fig 3: A basic groove formed upon dimerization and centered on K851 is responsible for single-stranded DNA/RNA binding.A, binding of the Ints3 C-terminal domain wild-type, mutant, and Ints6-807-887 complex protein with single-stranded DNA (30 mer consecutive thymines, dT30). The protein concentrations used were up to 200 μM. Representative images of EMSA are shown. B, quantification of (A) from three independent repeats. Error bars represent standard deviation. C, binding of the Ints3 C-terminal domain wild-type, mutant, and Ints6-807-887 complex protein with single-stranded RNA (30 mer consecutive uracils, rU30). The protein concentrations used were from 0 to 25 μM. Representative images of EMSA are shown. D, quantification of (C) from three independent repeats. Error bars represent standard deviation.

Fig 4: Overall structure of the human Ints3 C-terminal domain.A, domain organization of human Ints3. Ints3 contains an N-terminal SSB1/SSB1P1 binding domain (residues 1–500, PDB ID 4OWT), a C-terminal domain (residues 555–976) followed by a classical importin α/β pathway-dependent nuclear localization signal (cNLS), predicted by cNLS mapper (55). B, limited trypsin treatment generates two bands, Band II and III, from full-length C-terminal domain (Band I). N-terminal amino acid sequences of band II and III were obtained by Edman degradation method. C, the Ints3 C-terminal domain is an elongated HEAT-repeat α-solenoid consisting of 11 HEAT repeats (HR1-11). Each repeat is composed of an a-helix (cyan) and a b-helix (magenta), connected by a short linker. Helix 1a of HR1 was not modeled due to weak density. One confirmed trypsin cleavage site (between K914 and L915) lies within the HR10 intrarepeat loop where electron density for residues N899–A916 is missing.

Fig 5: Integrator complex regulates NELF-mediated RNAPII pausing at coding genes.(a) Venn diagram showing the intersection among differentially expressed (DE) genes identified by microarray analysis (n=3) upon depletion of −INTS11, −INTS3− or −NELF-E (see Methods; P<0.001). (b) Genomic view of ChIP-Seq data over the protein coding MTF2 gene for: H3K4me3, total RNAPII, NELF-E, SPT5, INTS3 and INTS11. (c) Heat maps showing the binding of NELF-E, RNAPII, INTS3 and INTS11 by ranking the top 10,000 genes according to NELF binding (+/− 250 bp from TSSs; see Methods). (d) Heat map showing the intersection analyses between DE (down- or up- regulated) genes and the corresponding TSS bound or not (+/−) by NELF-E, INTS3 and INTS11 (see Methods). Differential expression (DE) was analysed in R using limma (adjusted P<0.01; see Supplementary Dataset 2B–D; see Methods). The enrichment between these two lists is indicated in Log P-value (see colour bar). (e) Averaged RNA RNAPII profiles as measured by ChIP-Seq as a function of NELF-E-, Spt5-, INTS3 and INTS11 binding. The ChIP-Seq profiles of ‘High/Low pausing’ are indicated for comparison (RNAPII profiles of highly paused (blue line)- or active genes (light blue line), respectively, as previously described22). Genes bound by −NELF, SPT5 and INTS3 with or without INTS11 (purple and orange line, respectively) or by −NELF and SPT5 with or without INTS3 (yellow and orange line, respectively) or by SPT5, INTS3 and INTS11 (green line). (f) Box plots showing the RNAPII pausing indices depending on NELF/SPT5/INTS3 and INTS11 binding or not (+/−) to the corresponding TSS. P-values, Wilcoxon pair-wise test (see also Supplementary Fig. 7A).