Abstract
Integrator is a multi-subunit protein complex associated with RNA polymerase II (Pol II), with critical roles in noncoding RNA 3′-end processing and transcription attenuation of a broad collection of mRNAs. IntS11 is the endonuclease for RNA cleavage, as a part of the IntS4-IntS9-IntS11 Integrator cleavage module (ICM). Here we report a cryo-EM structure of the Drosophila ICM, at 2.74 Å resolution, revealing stable association of an inositol hexakisphosphate (IP6) molecule. The IP6 binding site is located in a highly electropositive pocket at an interface among all three subunits of ICM, 55 Å away from the IntS11 active site and generally conserved in other ICMs. We also confirmed IP6 association with the same site in human ICM. IP6 binding is not detected in ICM samples harboring mutations in this binding site. Such mutations or disruption of IP6 biosynthesis significantly reduced Integrator function in snRNA 3′-end processing and mRNA transcription attenuation. Our structural and functional studies reveal that IP6 is required for Integrator function in Drosophila, humans, and likely other organisms.
Similar content being viewed by others
Introduction
Integrator is a 15-subunit complex associated with RNA polymerase II (Pol II) that is crucial for 3′-end processing of snRNAs1 and other noncoding RNAs2,3,4,5, as well as transcription attenuation through cleavage of a broad set of nascent mRNAs6,7,8,9,10. The broad importance of Integrator function to gene regulation is evidenced by the wide range of human disease states attributed to dysfunction of its subunits11,12.
The Integrator subunits, named IntS1 through IntS15, can be purified as a complex but also form several sub-modules. For example, our earlier studies have shown that IntS4-IntS9-IntS11 forms the Integrator cleavage module (ICM)13,14. IntS9 and IntS11 are paralogs of CPSF100 and CPSF73 in the canonical and U7 replication-dependent histone pre-mRNA 3′-end processing machineries, and CPSF73 catalyzes the cleavage reaction in both machineries15,16,17. The subunits IntS10-IntS13-IntS14 form a putative nucleic acid binding module18,19, and the IntS5-IntS8 complex19,20 is critical for recruiting protein phosphatase 2 A (PP2A)20,21,22.
The structures of several Integrator components have been reported over the years, including the C-terminal domain (CTD2) complex of human IntS9-IntS1123, the N- and C-terminal domains of human IntS324,25, human IntS13-IntS14 complex18, and the human ICM19. In addition, the structures of human Integrator in complex with PP2A21 and Pol II26,27 were reported recently. These structures reveal how Integrator is organized overall. However, insight is still lacking as to how Integrator activity, especially that of the endonuclease IntS111, is regulated within this machinery and by other factors.
Here we report a cryo-EM structure of the Drosophila ICM at 2.74 Å resolution, which unexpectedly reveals the stable association of an inositol hexakisphosphate (IP6) molecule. We have also identified IP6 binding to the same site in the human ICM. Mutations in the IP6 binding site disrupt IP6 binding but not Integrator assembly. On the other hand, such mutations or disruption of IP6 biosynthesis significantly reduce Integrator function in snRNA 3′-end processing and mRNA transcription attenuation.
Results and discussion
Structure of Drosophila ICM
To gain structural insight into the inner workings of IntS11, we co-expressed and purified the Drosophila ICM using baculovirus-infected insect cells (Fig. 1a, b) and determined its structure at 2.74 Å resolution by cryo-EM (Figs. 1a, c, d, 2a–c, Table 1, Supplementary Fig. 1). The overall structure of Drosophila ICM (Fig. 1c, d) is generally similar to that of human ICM19,21 (Fig. 3a, b). IntS11 is in a closed, inactive state in our structure of Drosophila ICM, as well as in those reported recently of human ICM19,21.
The structure shows that the C-terminal segments of IntS9 and IntS11 contain two separate domains, CTD1 and CTD2 (Fig. 1a, c), similar to their paralogs CPSF100 and CPSF7317. The two CTD2 domains have weak EM density (Fig. 2a–c, Supplementary Fig. 1), and their atomic models were guided by the structure of the human IntS9-IntS11 CTD2 complex23. The CTDs have extensive interactions with each other, facilitating the association of IntS9 and IntS11.
The metallo-β-lactamase and β-CASP domains of IntS9 and IntS11 form a pseudo-dimer in this structure (Fig. 1c), remarkably similar to the pseudo-dimer for the equivalent domains of CPSF100 and CPSF73 in the active U7 machinery (Fig. 1e)17. The N-terminal domain (NTD) of IntS4 contacts the metallo-β-lactamase domain of IntS9 and the back face of IntS11 metallo-β-lactamase and β-CASP domains (Fig. 1c, d), which may promote the formation of this pseudo-dimer.
An unexpected IP6-binding site in Drosophila ICM
The structure of Drosophila ICM unexpectedly revealed the presence of an inositol hexakisphosphate (IP6) molecule (Fig. 1c, d), with good quality EM density (Fig. 4a). The compound was bound to the ICM during expression in insect cells and remained associated through two column purification steps, suggesting that it has a high affinity for ICM.
IP6 is located at an interface among all three subunits of the ICM, formed by the N-terminus and the linker to CTD1 of IntS9, the CTD1 of IntS11, and the first few helical repeats of IntS4 NTD (Fig. 4b). IP6 has ionic interactions with all three subunits, including Lys189 of IntS4, the N-terminal ammonium ion, Arg2, Arg504, Lys508 and Arg509 of IntS9, and Lys462 of IntS11 (Fig. 4b). These residues create a large, highly positively charged pocket (Fig. 4c), accommodating IP6 or other highly negatively charged molecules.
The EM density for IP6 is of sufficient quality such that we can recognize the 2-position of inositol (Fig. 4a). The phosphate at this position is in the axial position, whereas those at the other positions are equatorial. The 2-phosphate interacts with Arg504 and Lys508 of IntS9 (Fig. 4b), suggesting that IP5, which lacks this phosphate, would have weaker interactions with ICM. This is consistent with the observation that the EM density for all six phosphates is roughly equivalent (Fig. 4a), suggesting that they are present with similar occupancies. The structure suggests that a pyrophosphate group at the 1 position (1-IP7) could be accommodated, while accommodation of such a group at the 5 position (5-IP7) could be more difficult (Fig. 4c). Nonetheless, there is little indication of extra EM density at the 1 or the 5 position (Fig. 4a). Further studies are needed to ascertain whether inositol pyrophosphates play a role in Integrator function.
IP6 binding is also found in human ICM
Residues in the IP6 binding site are generally conserved among IntS9 and IntS11 homologs (Fig. 5a). While Lys189 is glutamine in vertebrate IntS4, IP6 should maintain favorable interactions with the dipoles of the helices in the IntS4 NTD.
In the structure of the human ICM19, EM density highly consistent with IP6 is present near the N-terminus of INTS9 as well (Fig. 5b), although IP6 was not included in that atomic model. The detailed binding mode of IP6 in human ICM has only slight differences compared to that in Drosophila ICM (Fig. 5c). In the structure of the human Integrator-PP2A complex (EMDB 30473)21, some EM density is present near the N-terminus of INTS9, although the quality of the density here is poor. In fact, a few N-terminal residues of INTS9 were built into this density. Overall, the structural observations suggest that this pocket is likely to bind IP6 or another negatively charged compound(s) and play a role in the function of ICM in general.
Mutations in IP6-binding site block IP6 binding
To characterize the importance of IP6 for Integrator, we first created mutations in the IP6 binding site that changed the positively charged residues to negatively charged ones and assessed their impact on IP6 binding and Integrator assembly. We developed a protocol that enabled us to confirm by mass spectrometry the binding of IP6 to wild-type human or Drosophila ICM samples purified from insect cell expression (Fig. 6a). We then produced both Drosophila and human ICM samples containing the K462E mutation in IntS11 and the human ICM sample containing the K510E/R511E double mutation in IntS9. The residue equivalent to Arg504 of Drosophila IntS9 is Ala in human IntS9 (Fig. 5a), and hence it was not mutated. The mutants produced gel filtration profiles that are comparable to those of wild-type ICMs (Fig. 6b, c), but we failed to detect IP6 in our mass spectrometry experiments on them (Supplementary Fig. 2).
We also created Drosophila DL1 nuclear extracts from cell lines stably expressing FLAG-IntS11-WT, FLAG-IntS11-K462E, FLAG-IntS9-WT, or FLAG-IntS9-R2E and then purified associated complexes using anti-FLAG affinity. We observed that the IntS11-K462E and the IntS9-R2E mutations reduced but did not completely abolish association with Integrator subunits (Fig. 6d, e). These data confirm the existence of the IP6 binding site in Drosophila and human Integrator and show that mutations of residues in this binding site can block IP6 binding but do not completely abolish Integrator assembly.
IP6 is required for Integrator function
To determine the functional significance of IP6 binding to Integrator, we devised a method to induce expression of IntS11-WT or IntS11-K462E in cells where endogenous IntS11 has been depleted using dsRNA targeting its 5′ and 3′ UTRs. We observed that treatment of DL1 cells with IntS11 dsRNA resulted in effective depletion of endogenous IntS11, whereas induction of IntS11 transgenes allowed expression of near endogenous levels of IntS11-WT or IntS11-K462E proteins (Fig. 7a). We then analyzed the impact of IntS11 depletion on U4snRNA processing, transcriptional attenuation of a previously validated mRNA-encoding gene called tigrin (tig)6, and a gene found not to be regulated by Integrator (Bj1). As expected, depletion of IntS11 resulted in a significant increase in U4snRNA misprocessing as well as tig transcription but did not affect Bj1 (Fig. 7b). Importantly, these phenotypes were restored upon re-expression of IntS11-WT but could not be rescued by the IntS11-K462E mutant (Fig. 7b). Similar observations were made with the R2E and the R504E/K508E/R209E triple mutation (trmt) in IntS9 (Fig. 7c, d). These results show that although these mutations do not completely abolish the assembly of Integrator subunits, they are significantly disruptive to Integrator function, underscoring the critical need for IP6 binding for Integrator activity.
Finally, we transfected cells with two different siRNAs targeting inositol polyphosphate multikinase (IPMK), an upstream kinase required for both IP5 and IP6 biosynthesis28,29. We assessed Integrator function using two reporters where the U7snRNA promoter, gene body, and 3′ cleavage site are placed upstream of either GFP or luciferase with the rationale that loss of Integrator function leads to transcriptional readthrough and expression of the downstream open reading frames (Fig. 8a). We observed that depletion of IPMK resulted in increased expression of GFP and luciferase relative to control (Fig. 8b, c). Notably, the siRNA capable of more significant IPMK depletion (#2 in Fig. 8b, c) produced much higher GFP/luciferase expression, in fact reaching a level similar to that observed after depletion of IntS11. These data further reveal the critical nature of IP6 in Integrator function and Pol II transcription.
The binding site for IP6 in ICM is located far (55 Å) from the active site of IntS11 and is on the opposite face from the opening for the canyon in IntS11 for binding RNA (Fig. 1c). Therefore, this binding site is unlikely to affect the catalysis by IntS11 directly. In addition, the IP6 binding site is far away from other modules of Integrator and Pol II in the reported structures (Supplementary Figs. 3a, b)21,26,27, and therefore IP6 likely exerts its effects on Integrator solely through the ICM. Mutation of Lys462 also affected human Integrator function, and it was suggested this binding site could interact with a part of the snRNA substrate19, although our functional studies have demonstrated a critical role for IP6 in Integrator activity. The binding of IP6 is also observed in structures of the spliceosome30,31, although the function of this binding has not been reported. Our observations expand the repertoire of IP6 as a molecule impacting RNA editing and processing32,33,34 and other processes in the cell35,36.
Methods
Protein expression and purification
Drosophila IntS4, IntS9, and IntS11 were co-expressed in insect cells. IntS9 and IntS11 were cloned into the pFL acceptor vector. N-terminal 6xHis-tagged IntS4 was cloned into the pSPL donor vector. These two vectors were fused by Cre recombinase. Tni insect cells (Expression Systems) (2 × 106 cells·ml–1) were infected with 16 ml of IntS4-IntS9-IntS11 P2 virus and harvested after 48 h.
For purification, the cell pellet was resuspended and lysed by sonication in 100 ml of buffer containing 20 mM Tris (pH 8.0), 250 mM NaCl, 2 mM βME, 5% (v/v) glycerol, and one tablet of protease inhibitor mixture (Sigma). The cell lysate was then centrifuged at 15,000 × g for 40 min at 4 °C. The protein complex was purified from the supernatant via nickel affinity chromatography (Qiagen). The protein complex was further purified using a Hiload 16/60 Superdex 200 column (Cytiva). The IntS4-IntS9-IntS11 complex was concentrated to 2 mg·ml–1 in a buffer containing 20 mM Tris (pH 8.0), 300 mM NaCl, and 2 mM DTT, and stored at −80 °C.
EM specimen preparation and data collection
All specimens for cryo-EM were frozen with an EM GP2 plunge freezer (Leica) set at 20 °C and 99% humidity. Cryo-EM imaging was performed in the Simons Electron Microscopy Center at the New York Structural Biology Center using Leginon37.
For the IntS4-IntS9-IntS11 complex, a 3.5 μL aliquot at 0.1 mg·ml–1 was applied to one side of a Quantifoil 400 mesh 1.2/1.3 gold grid with graphene oxide support film (Quantifoil). After 30 s, the grid was blotted for 1.5 s on the other side and plunged into liquid ethane. 3083 image stacks were collected on a Titan Krios electron microscope at New York Structural Biology Center, equipped with a K3 direct electron detector (Gatan) at 300 kV with a total dose of 51 e− Å−2 subdivided into 40 frames in 2 s exposure using Leginon. The images were recorded at a nominal magnification of 81,000× and a calibrated pixel size of 1.083 Å, with a defocus range from –1 to −2.5 μm.
Image processing
Image stacks were motion-corrected and dose-weighted using RELION 3.138. The patch CTF parameters were determined with cryoSPARC39. First, 2,920,144 particles were auto-picked and subjected to 2D classification in cryoSPARC. 1,149,821 particles in classes with recognizable features by visual inspection were used to generate eight 3D initial models by ab initio reconstruction. After one round of heterogeneous refinement, 620,438 particles were imported to RELION for CTF refinement and Bayesian polishing, yielding a map at 2.74 Å resolution. An analysis of the map gave sphericity of 0.96340, suggesting that anisotropy is not a problem for this reconstruction.
Model building
Atomic models for IntS11, IntS9, and IntS4 were built manually into the cryo-EM density with Coot41. Homology models for Drosophila IntS9 and IntS11 were generated with I-TASSER42, based on the structures of human CPSF100 and CPSF7343. The atomic models were improved by real-space refinement with the program PHENIX44.
The model of IP6 in the Drosophila ICM was placed in the human ICM cryo-EM density (EMDB 12159)19 and manually adjusted to fit the density. Real-space refinement was then used to optimize the fitting of IP6 to the EM density.
LC-MS/MS analysis of IP6 binding
Samples were diluted 100x in 20% (v/v) acetonitrile to help denature the complex and reduce the salt concentration, after which they were placed into clean autosampler tubes and analyzed via LC-MS/MS.
IP6 analysis was carried out on a Dionex Ultimate 3000 UHPLC coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher). IP6 was separated on an Accucore aQ C18 2.1 ×150 mm column (Thermo Fisher). The mobile phases were A: 20 mM ammonium acetate, pH 9.0, and B: acetonitrile. The flow rate was set to 400 μL/min, and the column oven was set to 25 °C. 40 μL of each sample was injected, and the analytes were eluted isocratically at 20% B. The gradient was then ramped to 50% B to wash the column and returned to starting conditions for re-equilibration. The total runtime was 4.5 min.
The Q Exactive Plus was operated in negative mode with a heated electrospray ionization (HESI) source, in conjunction with a parallel reaction monitoring (PRM) method. An IP6 standard was used to determine the [M-H] mass (658.8543) and [M-2H] mass (328.9238), both of which were isolated and fragmented to increase the confidence of IP6 identification. The standard was also used to determine fragment ions, and retention times, and to optimize the collision energy. Precursor ions were isolated with a 2.0 m/z isolation width and then fragmented in the collision cell with a collision energy of 30. The maximum injection time was set to 100 ms, while the AGC target was set to 2e5. The resulting fragment ions were detected in the Orbitrap with a resolution of 17,500 at m/z 200. Fragment ions 560.8766 m/z [M − H] and 480.9104 m/z [M − 2H] were used to identify the analytes. Chromatograms were extracted with a 10 ppm mass error, and peaks were integrated using XCalibur software (Thermo Fisher).
Plasmid construction and stable cell lines generation
For mutation analysis of Drosophila IntS9 and IntS11, site-directed PCR mutagenesis was used to create the IntS9-R2E, IntS9-trmt, and IntS11-K462E mutations (primer sequences are in Supplementary Table 1). Wild-types and the mutants of dIntS9 and IntS11 were subsequently cloned into the pMT-3xFLAG-puro vector6 to express in DL1 cells inducibly. All plasmids were sequenced to confirm identity. To generate cells stably expressing the FLAG-IntS9-WT, FLAG-IntS9-R2E, FLAG-IntS9-trmt, FLAG-IntS11-WT, FLAG-IntS11-K462E, and eGFP control transgenes, 2×106 cells were first plated in regular maintenance media in a 6-well dish overnight. Two micrograms of expressing plasmids were transfected using Fugene HD (Promega, #E2311). After 24 h, 2.5 μg/mL puromycin was added to the media to select and maintain the cell population.
Nuclear extract preparation
Five 150 mm dishes of each condition of confluent cells (pretreated with 500 μM CuSO4 for 24 h) were collected and washed in cold PBS before being resuspended in ten times volumes of the cell pellet of Buffer A (10 mM Tris pH 8, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.2 mM PMSF). Resuspended cells were allowed to swell during a 15 min rotation at 4 °C. After pelleting down at 1000 × g for 10 min, two times volumes of the original cell pellet of Buffer A were added, and cells were homogenized with a dounce pestle B for 40 strokes on ice. Nuclear and cytosolic fractions were separated by centrifuging at 800 × g for 10 min. To attain a nuclear fraction, the pellet was washed once with Buffer A before being resuspended in two times volumes of the original cell pellet of Buffer C (20 mM Tris pH 8, 420 mM NaCl, 1.5 mM MgCl2, 25% (v/v) glycerol, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT). The samples were then homogenized with a dounce pestle B for 20 strokes on ice and rotated for 30 min at 4 °C before centrifuging at 15,000 × g for 30 min at 4 °C. Finally, supernatants were collected and subjected to dialysis in Buffer D (20 mM HEPES, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 20% (v/v) glycerol) overnight at 4 °C against a 3.5 kDa MWCO membrane (Spectrum Laboratories, #132720). Prior to any downstream applications, nuclear extracts were centrifuged again at 15,000 × g for 3 min at 4 °C to remove any precipitate.
Western blotting and anti-FLAG affinity purification
To check protein expression, cells were lysed directly in wells in 2× SDS sample buffer (120 mM Tris pH 6.8, 4% SDS, 200 mM DTT, 20% (v/v) glycerol, and 0.02% bromophenol blue). Lysates were incubated at room temperature with periodic swirling before a 10 min boiling at 95 °C and a short sonication. Denatured protein samples were then resolved in a 10% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, #1620177). Blots were probed by custom-designed Drosophila antibodies as previously described6 diluted in PBS-0.1% Tween supplemented with 5% nonfat milk. To detect proteins from 293 T lysate, anti-hInts11 (Bethyl, #A301-274A), anti-hIMPK (Thermo, #PA5-21629), anti-GFP (Clontech, #632381), anti-alpha Tubulin (abcam, #ab15246), and anti-GAPDH (Thermo, #MA5-15738) were used at the dilution suggested by the manufacturer.
To purify FLAG-tagged Integrator complexes, 1 mg of nuclear extract was mixed with 40 μL anti-Flag M2 affinity agarose slurry (Sigma, #A2220) equilibrated in binding buffer (20 mM HEPES pH 7.4, 100 mM KCl, 10% (v/v) glycerol, 0.1% NP-40) and rotated for 2 h at 4 °C. Following the 2 h incubation/rotation, five sequential washes were carried out in binding buffer with a 10 min rotation at 4 °C followed by a 500 × g centrifugation at 4 °C. After the final wash, the binding buffer supernatant was removed using a pipette, and the protein complexes were eluted from the anti-FLAG resin by adding 40 μL of 2× sample buffer and boiled at 95 °C for 5 min. For input samples, nuclear extracts were mixed with 5× loading buffer and boiled, and 1/10 volume of the immunoprecipitation reaction was loaded on SDS-PAGE.
RNA Interference
Double-stranded RNAs targeting the 5′ and 3′ UTRs of Drosophila IntS11 and an RNAi-resistant region of the IntS9 constructs were generated by in vitro transcription of PCR templates containing the T7 promoter sequence on both ends using MEGAscript kit (Thermo, #AMB13345). For RNA interference experiments, 1.5 × 106/ml of DL1 cells were washed into serum-free media and seeded into a 6-well plate along with 10 μg of dsRNA. After a 1 h incubation, 2 ml of complete growth medium was added, followed by 60 h of incubation before harvest. To perform rescue experiments while knocking down, cells were also treated with 100 μM CuSO4 throughout the 60 h incubation period to induce expression of the RNAi-resistant FLAG-IntS9-WT, FLAG-IntS9-R2E, FLAG-IntS9-trmt, FLAG-IntS11-WT, FLAG-IntS11-K462E transgenes.
IntS11 (Sigma, #SASI_Hs01_00032429), IPMK (Sigma, #SASI_Hs01_00047017, top strand sequence CAAACGAUUUAUACCUAAA[dT][dT], and #SASI_Hs01_00047015, GGUUUAUGCUGCUGACUGU[dT][dT]), and control (Sigma, #SIC002) siRNAs (2 μL each of 20 mM stock) were incubated in 50 μL of prewarmed (room temperature) Opti-MEM I reduced serum medium (GIBCO) for 5 min at room temperature. Similarly, RNAiMax (2 μL per well) was incubated in 50 μL prewarmed Opti-MEM I reduced serum medium for 5 min. The siRNA and RNAiMAX dilutions were mixed and incubated for 20 min at room temperature. 2 × 105 293 T cells were seeded into a 24-well plate, and the prepared transfection mixes of 40 pmols siRNAs were added to each well. Cells were transfected with 60 pmols of siRNA a second time after 24 h of incubation. The cells were expanded into 12-well plate after a total of 48 h and harvested at a total of 72 h of incubation under standard mammalian cell culture conditions.
Reporter cell lines establishment and luciferase measurement
To construct a reporter plasmid, pAAVS1-TLR targeting vector (Addgene, #64215) was cut with CalI and PspXI to substitute with U11/U7 small nuclear RNA (500 bp upstream of transcription start site, coding region, and 50 bp downstream of coding region) followed by Renilla luciferase/GFP coding region and SV40 poly(A) signal. The reporter plasmid was co-transfected with the gRNA cloned into pU6-(BbsI) CBh-Cas9-T2A-mCherry (Addgene, #64324), targeting AAVS1 locus into 293 T. Briefly, an equal amount of plasmids were transfected with lipofectamine 2000 for 24 h before selecting with 800 ng/ml puromycin for 2 days. Cells were grown in a regular medium without puromycin for a week before clonal selection.
Renilla Luciferase Assay System (Promega, #E2810) was used to assess luciferase activity in the clonal reporter cell lines. The line with the highest luciferase activity after hIntS11 knockdown was selected. To test the role of IPMK in Integrator’s function, the reporter cell lines were seeded in 24-well plate and knocked down twice with siRNAs as described. Luciferase activity was measured according to the manufacturer’s protocol. The background luciferase activity of each sample was calculated by interpolation and subtraction. Briefly, scatterplot and trendline were plotted by protein quantity versus luciferase activity obtained from the same amount of reporter cell line lysate, and an increased amount of the lysate was measured. The bars represent the average of triplicate biological repeats.
RT-qPCR quantification and analysis
Data were analyzed using the ΔΔCt method with Rps17 as the reference gene and LacZ dsRNA-treated cells as the control, and all PCR amplicon primers were described previously45.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
The structure of the Drosophila ICM-IP6 complex has been deposited at the PDB under accession code 7SN8. The cryo-EM map of the Drosophila ICM-IP6 complex has been deposited at the EMDB under accession code 25214. Source data are provided with this paper.
References
Baillat, D. et al. Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell 123, 265–276 (2005).
Baillat, D. & Wagner, E. J. Integrator: surprisingly diverse functions in gene expression. Trends Biochem. Sci. 40, 257–264 (2015).
Mendoza-Figueroa, M. S., Tatomer, D. C. & Wilusz, J. E. The Integrator complex in transcription and development. Trends Biochem. Sci. 45, 923–934 (2020).
Kirstein, N., Gomes Dos Santos, H., Blumenthal, E. & Shiekhattar, R. The Integrator complex at the crossroad of coding and noncoding RNA. Curr. Opin. Cell Biol. 70, 37–43 (2020).
Beltran, T., Pahita, E., Ghosh, S., Lenhard, B. & Sarkies, P. Integrator is recruited to promoter-proximally paused RNA Pol II to generate Caenorhabditis elegans piRNA precursors. EMBO J. 40, e105564 (2020).
Elrod, N. D. et al. The Integrator complex attenuates promoter-proximal transcription at protein-coding genes. Mol. Cell 76, 738–752.e7 (2019).
Tatomer, D. C. et al. The Integrator complex cleaves nascent mRNAs to attenuate transcription. Genes Dev. 33, 1525–1538 (2019).
Beckedorff, F. et al. The human integrator complex facilitates transcriptional elongation by endonucleolytic cleavage of nascent transcripts. Cell Rep. 32, 107917 (2020).
Thomas, Q. A. et al. Transcript isoform sequencing reveals widespread promoter-proximal transcriptional termination in Arabidopsis. Nat. Commun. 11, 2589 (2020).
Rosa-Mercado, N. A. et al. Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression. Mol. Cell 81, 502–513.e4 (2021).
Federico, A. et al. Pan-cancer mutational and transcriptional analysis of the integrator complex. Int. J. Mol. Sci. 18, 936 (2017).
Krall, M. et al. Biallelic sequence variants in INTS1 in patients with developmental delays, cataracts, and craniofacial anomalies. Eur. J. Hum. Genet. 27, 582–593 (2019).
Albrecht, T. R. & Wagner, E. J. snRNA 3’ end formation rquires heterodimeric association of Integrator subunits. Mol. Cell. Biol. 32, 1112–1123 (2012).
Albrecht, T. R. et al. Integrator subunit 4 is a ‘Symplekin-like’ scaffold that associates with INTS9/11 to form the Integrator cleavage module. Nucleic Acids Res. 46, 4241–4255 (2018).
Mandel, C. R. et al. Polyadenylation factor CPSF-73 is the pre-mRNA 3’-end-processing endonuclease. Nature 444, 953–956 (2006).
Dominski, Z., Yang, X.-C. & Marzluff, W. F. The polyadenylation factor CPSF-73 is involved in histone-pre-mRNA processing. Cell 123, 37–48 (2005).
Sun, Y. et al. Structure of an active human histone pre-mRNA 3’-end processing machinery. Science 367, 700–703 (2020).
Sabath, K. et al. INTS10-INTS13-INTS14 form a functional module of Integrator that binds nucleic acids and the cleavage module. Nat. Commun. 11, 3422 (2020).
Pfleiderer, M. M. & Galej, W. P. Structure of the catalytic core of the Integrator complex. Mol. Cell 81, 1246–1259 (2021).
Huang, K. L. et al. Integrator recruits protein phosphatase 2A to prevent pause release and facilitate transcription termination. Mol. Cell 80, 345–358.e9 (2020).
Zheng, H. et al. Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase. Science 370, eabb5872 (2020).
Vervoort, S. J. et al. The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer. Cell 184, 3143–3162.e32 (2021).
Wu, Y., Albrecht, T. R., Baillat, D., Wagner, E. J. & Tong, L. Molecular basis for the interaction between Integrator subunits IntS9 and IntS11 and its functional importance. Proc. Natl Acad. Sci. USA 114, 4394–4399 (2017).
Ren, W. et al. Structural basis of SOSS1 complex assembly and recognition of ssDNA. Cell Rep. 6, 982–991 (2014).
Li, J. et al. Structural basis for multifunctional roles of human Ints3 C-terminal domain. J. Biol. Chem. 296, 100112 (2021).
Fianu, I. et al. Structural basis of Integrator-mediated transcription regulation. Science 374, 883–887 (2021).
Zheng, H. et al. Structural basis of INTAC-regulated transcription. bioRxiv. https://www.biorxiv.org/content/10.1101/2021.11.29.470345v1 (2021).
Seeds, A. M., Sandquist, J. C., Spana, E. P. & York, J. D. A molecular basis for inositol polyphosphate synthesis in Drosophila melanogaster. J. Biol. Chem. 279, 47222–32 (2004).
Lee, B., Park, S. J., Hong, S., Kim, K. & Kim, S. Inositol polyphosphate multikinase signaling: multifaceted functions in health and disease. Mol. Cells 44, 187–194 (2021).
Fica, S. M. et al. Structure of a spliceosome remodelled for exon ligation. Nature 542, 377–380 (2017).
Zhang, X. et al. An atomic structure of the human spliceosome. Cell 169, 918–929.e14 (2017).
Macbeth, M. R. et al. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309, 1534–9 (2005).
Montpetit, B. et al. A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export. Nature 472, 238–42 (2011).
Gat, Y. et al. InsP(6) binding to PIKK kinases revealed by the cryo-EM structure of an SMG1-SMG8-SMG9 complex. Nat. Struct. Mol. Biol. 26, 1089–1093 (2019).
Blind, R. D. Structural analyses of inositol phosphate second messengers bound to signaling effector proteins. Adv. Biol. Regul. 75, 100667 (2020).
Lee, S., Kim, M. G., Ahn, H. & Kim, S. Inositol pyrophosphates: signaling molecules with pleiotropic actions in mammals. Molecules 25, 2208 (2020).
Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).
Emsley, P. & Cowtan, K. D. Coot: model-building tools for molecular graphics. Acta Cryst. D60, 2126–2132 (2004).
Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738 (2010).
Zhang, Y., Sun, Y., Shi, Y., Walz, T. & Tong, L. Structural insights into the human pre-mRNA 3’-end processing machinery. Mol. Cell 77, 800–809 (2020).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D. Struct. Biol. 75, 861–877 (2019).
Ezzeddine, N. et al. A subset of Drosophila integrator proteins is essential for efficient U7 snRNA and spliceosomal snRNA 3’-end formation. Mol. Cell. Biol. 31, 328–341 (2011).
Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).
Acknowledgements
We thank the staff at the Columbia University Cryo-Electron Microscopy Center for help with screening EM grids, Huihui Kuang and the staff at the New York Structural Biology Center for help with cryo-EM data collection. This research is supported by the Cancer Prevention Research Institute of Texas (CPRIT) grant RP170593 (to K.-L.H.), a Kempner Predoctoral Fellowship (to N.D.E.), NIH grants R01GM134539 (to E.J.W.), and R35GM118093 (to L.T.).
Author information
Authors and Affiliations
Contributions
N.D.E., M.K.J., and K.-L.H. conducted biochemical purifications, immune-precipitations, and western blotting; M.-H.L. and L.T. conducted all structural analyses; N.D.E. and M.K.J. generated all constructs used in DL1/293T cells; K.W. performed all mass spectrometry; L.T. and E.J.W. conceived the project and wrote the manuscript, with comments from all authors.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Lin, MH., Jensen, M.K., Elrod, N.D. et al. Inositol hexakisphosphate is required for Integrator function. Nat Commun 13, 5742 (2022). https://doi.org/10.1038/s41467-022-33506-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-022-33506-3
This article is cited by
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.