Functional mechanisms of specific proteins of pre-catalytic B complex spliceosomes in constitutive and alternative splicing
Final Report Abstract
The spliceosome is a large and dynamic macromolecular machine composed of many RNA and protein subunits. It is responsible for the removal of non-coding intervening sequences (introns) from precursor messenger RNAs (pre-mRNAs) and concomitant ligation of the coding sequences (exons) to produce mature mRNAs for protein biosynthesis on the ribosome. This so-called pre-mRNA splicing is an essential step in the expression of the majority of metazoan protein-coding genes. Moreover, metazoan pre-mRNAs typically contain more than one intron and can be alternatively spliced, giving rise to more than one mature mRNA, and thus more than one protein, originating from the same gene. A hallmark of the spliceosome is its stepwise assembly, catalytic activation, splicing catalysis and disassembly, which occurs anew for each splicing reaction. In the course of its duty cycle, the composition and structure of the spliceosome are repeatedly remodeled. The molecular mechanisms underlying constitutive and alternative splicing are far from fully understood. In particular, the specific roles of many spliceosomal protein components remain unresolved. In this project, we have investigated the structures, interactions and functions of a group of so-called B-specific proteins, which join the spliceosome at the pre-catalytic stage and are expelled again during the following activation phase. Thus, they are present during a stage of splicing, when alternative splicing decisions can still be made. Moreover, several of these B- specific proteins are only found in spliceosomes of higher organisms, which also exhibit a higher degree of alternative splicing. However, at the beginning of this project, the precise roles of B-specific proteins during constitutive or alternative splicing were unknown. We elucidated interaction networks that involve spliceosomal B-specific proteins using high-throughput interaction screens and validated putative interactions using recombinant B-specific proteins in vitro. Based on these results, we elucidated crystal structures of several complexes of B-specific proteins, which allowed the design of specific protein variants that failed to support these interactions. In addition, structure-based biochemical and biophysical analyses revealed novel fundamental principles of protein-protein interactions, such as the fine-tuning of the interaction strength between a folded protein and an intrinsically unstructured region of a binding partner, due to the tendency of the intrinsically unstructured region to adopt binding-competent conformations in isolation. The structure of one subcomplex of B-specific proteins also revealed unexpected similarities to transcriptional corepressor complexes, suggesting novel functional links of splicing to chromatin organization and transcription via B-specific proteins. Our 3D electron cryo-microscopic structure of a pre-catalytic human spliceosome revealed the organization of B-specific proteins in a functional context. In agreement with our biochemical analyses using isolated components, the molecular organization of the B- specific proteins in the pre-catalytic spliceosome suggests that they are involved in (i) negatively regulating a key motor protein of the spliceosome, which is required for spliceosome activation, (ii) properly positioning part of the spliceosome's RNA network and (iii) stabilizing the structure of the pre-catalytic spliceosome. Biochemical investigations further clarified the role of the B-specific proteins during the assembly of an active spliceosome. Unlike previously thought, we found that the B-specific proteins are not required for stable integration of a major spliceosomal subunit (the U4/U6.U5 tri-snRNP) during the formation of a pre-catalytic spliceosome. Instead, based on (i) highthroughput RNA sequencing analyses in combination with knockdown of selected B-specific proteins and (ii) analysis of splicing in nuclear extracts using specifically engineered substrate pre-mRNAs in combination with protein depletion/add-back experiments, we showed that certain B-specific proteins are important for the efficient conversion of a precatalytic to an activated spliceosome, presumably by facilitating global conformational changes that are required during this step. Taken together, our work in the course of this project provided major new insights into the molecular structures, interactions and functions of a large group of splicing factors, the B- specific proteins. Our results clarified the molecular requirements for the efficient formation of a pre-catalytic spliceosome and revealed regulatory functions of B-specific proteins during spliceosome activation.
Publications
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(2015) SnapShot: Spliceosome dynamics I, II & III. Cell 161, 1474- 1474.e.1.; Cell 162, 456-456.e.1.; Cell 162, 690-690.e.1
Wahl MC, Lührmann R
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(2015) Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5′ splice site. RNA 21, 1993-2005
Boesler C, Rigo N, Agafonov DE, Kastner B, Urlaub H, Will CL, Lührmann R
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(2016) A spliceosome intermediate with loosely associated trisnRNP accumulates in the absence of Prp28 ATPase activity. Nat Commun 7, 11997
Boesler C, Rigo N, Anokhina MM, Tauchert MJ, Agafonov DE, Kastner B, Urlaub H, Ficner R, Will CL, Lührmann R
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(2016) Scaffolding in the spliceosome via single α-helices. Structure 24, 1972-1983
Ulrich A, Seeger M, Schütze T, Bartlick N, Wahl MC
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(2016) Structural basis for the functional coupling of the alternative splicing factors Smu1 and RED. Structure 24, 762-773
Ulrich AKC, Schulz JF, Kamprad A, Schütze T, Wahl MC
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(2016). Multiple protein-protein interactions converging on Prp38 during activation of the human spliceosome. RNA 22, 265-277
Schütze T, Ulrich AKC, Apelt L, Will CL, Bartlick N, Seeger M, Weber G, Lührmann R, Stelzl U, Wahl MC
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(2017) A new role for FBP21 as regulator of Brr2 helicase activity. Nucleic Acids Res, 13, 7922-7937
Henning LM, Santos KF, Sticht J, Jehle S, Lee CT, Wittwer M, Urlaub H, Stelzl U, Wahl MC, Freund C
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(2017) Cryo-EM structure of a pre-catalytic human spliceosome primed for activation. Cell 170, 701-713
Bertram K, Agafonov DE, Dybkov O, Haselbach D, Leelaram MN, Will CL, Urlaub H, Kastner B, Lührmann R, Stark H
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(2017) Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor. BMC Evol Biol 17, 91
Ulrich A, Wahl MC
