Role of the activating signal co-integrator complex in the regulation of gene expression

Prof. Markus C. Wahl, PhD

October 16, 2022

Our lab is interested in the molecular mechanisms and principles of regulation of gene expression processes that we study using integrative structural biochemical and multi-omics approaches.

Eukaryotic replication, genome maintenance, gene expression and co/post-transcriptional gene regulation are functionally coupled in space and time. One principle of coupling relies on moonlighting proteins that exert functions in multiple processes. Functional coupling in this case could involve the hierarchical servicing of different processes dependent upon, e.g., changes in the production, degradation or sub-cellular localization of such moonlighting factors. We propose to study this principle further, taking the activating signal co-integrator complex (ASCC) as an initial example.

ASCC (core subunits ASCC1, ASCC2 and ASCC3) has originally been identified as a co-activator of nuclear receptors and has subsequently been implicated in diverse transcription regulation events, including transcriptional responses to UV-induced DNA damage and viral infections. Furthermore, ASCC is involved in DNA alkylation damage repair, ribosome quality control and non-functional 18S ribosomal RNA decay. In this PhD project, we propose to further explore the molecular mechanisms underlying ASCC’s multi-functionality by using a multi-pronged approach, initially prioritizing ASCC’s roles in transcription regulation. Cells expressing tagged ASCC subunits will be exposed, directly or after knock-down/targeted degradation of endogenous subunits, to diverse stimuli that elicit ASCC-related transcriptional responses. Transcriptome sequencing, chromatin immunoprecipitation/sequencing, cross-linking and analysis of cDNAs, pull-down/mass spectrometry and proximity labelling/mass spectrometry will then be employed to delineate effects of individual subunits on gene expression and to identify process-specific auxiliary factors. After validation, newly identified ASCC interactors will be produced recombinantly to reconstitute ASCC in complex with process-specific auxiliary factors. Cross-linking/mass spectrometry and cryogenic electron microscopy will be used to derive the overall architectures and atomic-level 3D structures of the complexes. The structural information will serve to identify residues/regions essential for enzymatic functions, regulation or interactions of subunits. Dysfunctional variants, in which these residues/regions have been altered, will ultimately serve to establish cause-effect relationships between ASCC subunits, auxiliary factors and condition-dependent transcriptional responses.

In the past, we have produced and purified recombinant versions of all human ASCC components and of some known auxiliary proteins, tested binary and higher-order interactions in vitro and in cell culture, elucidated structures of ASCC sub-complexes (by using macromolecular crystallography, cryogenic electron microscopy and cross-linking/mass spectrometry) and tested functional consequences of observed interactions in a structure-guided manner 1,2 (Figure 1). Since a long time, we are also exploring the inner workings and modes of regulation of the close ASCC3 homolog, SNRNP200 3-5, a key spliceosome remodeling factor. E.g., recently and in close collaboration with the groups of Tugce Aktas, Christian Freund and Florian Heyd within this IMPRS, we have elucidated the structures of SNRNP200 in complex with intrinsically disordered constitutive splicing factors, alternative splicing factors and snRNP assembly factors, and have interrogated the functional consequences of these interactions in a manner as planned for the study of ASCC 6-8.

For more information have a look at the website of the Structural Biochemistry group



Figure 1. Interactions of SNRNP200 and ASCC3 with modulatory factors. (A) CryoEM structure of the SNRNP200 helicase region (SNRNP200HR; semi-transparent surface; NC, light grey; CC, blue) in complex with PRPF8Jab1 (brown) and the intrinsically disordered snRNP assembly chaperone, TSSC4 8. (B) CryoEM structure of ASCC3HR (semi-transparent surface; NC, light grey; CC, blue) in complex with ASC1 (red) 2. (C) SDS-PAGE of size exclusion chromatography elution fractions monitoring the interaction of ASCC3HR and different regions of ASC1 2. ASC1 constructs containing the ZnF (1-230; 152-581; 152-230) stably bind ASCC3HR, while a construct lacking the ZnF but containing the linker and the ASCH domain (281-581) does not. (D) Model of ASCC3HR-ASC1 bound to DNA (based on SNRNP200-U4 snRNA in a spliceosomal B complex [PDB ID 6AHD]). Rotation symbols, views relative to (B), left. (E) DNA-stimulated ATPase activity of ASCC3 constructs alone or in complex with ASC1 (indicated at the bottom). ASCC3HR constructs, in which either the NC (D611A) or the CC (D1463A) are inactivated show ATPase activity, and ASC1 does not significantly enhance the ASCC3HR ATPase. Values represent means ± SE; n = 3. Significance compared to wild type (wt) ASCC3HR; ns, not significant; ****, p ≤ 0.0001. (F) Stopped-flow/fluorescence-based DNA unwinding assays, showing that ASC1, but not ASC11-230, stimulates ASCC3HR helicase activity 2. (G) As (F), monitoring unwinding by ASCC3HR constructs, in which either the NC (D611A) or the CC (D1463A) are inactivated 2. Both NC and CC seem to act as helicases. The CC is a weak helicase in context of a dead NC, which is moderately stimulated by ASC1. The NC in context of a dead CC is more weakly activated by ASC1 than wt ASCC3HR or the CC in context of a dead NC.



1.   Jia J, Absmeier E, Holton N, Pietrzyk-Brzezinska AJ, Hackert P, Bohnsack KE, Bohnsack MT, Wahl MC (2020) The interaction of DNA repair factors ASCC2 and ASCC3 is affected by somatic cancer mutations. Nat Commun11, 5535.

2.   Jia J, Hilal T, Bohnsack KE, Chernev A, Tsao N, Schwarz J, Arumugam A, Parmely L, Holton N, Loll B, Mosammaparast N, Bohnsack MT, Urlaub H, Wahl MC (2022) Extended DNA threading through a dual-engine motor module in the activating signal co-integrator complex. Research Square - In Review,

3.   Santos KF, Mozaffari-Jovin S, Weber G, Pena V, Luhrmann R, Wahl MC (2012) Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome. Proc Natl Acad Sci U S A 109, 17418-17423.

4.   Mozaffari-Jovin S, Wandersleben T, Santos KF, Will CL, Luhrmann R, Wahl MC (2013) Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8. Science 341, 80-84.

5.   Absmeier E, Wollenhaupt J, Mozaffari-Jovin S, Becke C, Lee CT, Preussner M, Heyd F, Urlaub H, Luhrmann R, Santos KF, Wahl MC (2015) The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation. Genes Dev 29, 2576-2587.

6.   Henning LM, Santos KF, Sticht J, Jehle S, Lee CT, Wittwer M, Urlaub H, Stelzl U, Wahl MC, Freund C (2017) A new role for FBP21 as regulator of Brr2 helicase activity. Nucleic Acids Res 45, 7922-7937.

7.   Bergfort A, Preussner M, Kuropka B, Ilik IA, Hilal T, Weber G, Freund C, Aktas T, Heyd F, Wahl MC (2022) A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing. Nat Commun 13, 1132.

8.   Bergfort A, Hilal T, Kuropka B, Ilik IA, Weber G, Aktas T, Freund C, Wahl MC (2022) The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling. Nucleic Acids Res 50, 2938-2958.



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