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Introduction

Intron removal from premature-messenger RNA (pre-mRNA splicing) is a key step of gene expression and regulation. The spliceosome (SPL) promotes pre-mRNA splicing to produce non-coding RNAs and mature mRNA [1]. The SPL consists of 5 small nuclear (sn)RNAs and over 100 proteins. Almost 50% of the human SPL proteins are predicted to be intrinsically disordered (IDP) or to contain ID regions (IDR). These are highly adaptable and can promiscuously bind to different partners [2]. This feature enables their fast association/dissociation within the dynamic the Ribonucleoprotein (RNP)-assemblies of SPL. Among the SPL proteins containing IDR is SF3B1, which exhibits an IDR N-terminus domain (NTD) and a C-terminal domain (CTD) composed of 22 HEAT-repeats [3]. SF3B1 is entailed in the recognition of a key premRNA signaling sequence (branch point sequence (BPS)) and is the most frequently mutated SPL protein. Indeed, its cancer-associated mutations cause aberrant splicing by altering the BPS selection. SF3B1 is also object of post-translational modifications both in its NTD and CTD [4]. It was recently discovered that phosphorylation of SF3B1 at its NTD (NTDSF3B1) regulates splicing [5]. Cryo-electron microscopy (cryo-EM) recently supplied several near-atomic level resolution structures of the SPL, enormously contributing to advancing our understanding of its structural biology. Yet, the success of cryo-EM methods and X-ray crystallography remains limited to structured proteins/regions, while the characterization of IDP/IDRs severely lags behind. Remarkably, addressing the structure of IDR/IDPs remains out of reach even to the Artificial Intelligence algorithms that have recently revolutionized the structural biology field [6].

To unlock the functional role of NTD-SF3B1 we exploit the expertise of the Dr. Magistrato’s (PI) lab the only group that has performed to date all-atom MD simulations of the whole SPL complex in different states [7, 8] and that has characterized distinct functional aspects of the splicing mechanism [1]. The PI’s lab will also take advantage of the expertise of Dr. Onesti and Dr. Napolitano at ELETTRA Synchrotron Trieste, who will perform and train a CNR-IOM unit of personnel to purify the NTD-SF3B1 fragment in its unphosphorylated and phosphorylated forms and to carry out SAXS experiments. Additionally, the project will leverage the expertise of Prof. Pierattelli’s lab at the Magnetic Resonance Center (CERM) of the University of Florence, (secondary research unit), which has a long experience in the characterization of IDPs and their interactions by NMR spectroscopy [9-11]. They will also provide competence for the preparation of samples isotopically enriched for NMR spectroscopy.

References:

J. Borisek, L. Casalino, A. Saltalamacchia, S.G. Mays, L. Malcovati, A. Magistrato Acc. Chem. Res. 2020 54, 144
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L Casalino, G Palermo, A Spinello, U Rothlisberger, A Magistrato Proc. Natl. Acad. Sci. USA 2018 115, 6584
J. Borišek, A. Saltalamacchia, A. Gallì, G. Palermo, E. Molteni, L. Malcovati, A. Magistrato, Biomolecules 2019 9 (10), 633
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S. Contreras-Martos, A. Piai, S. Kosol, M. Varadi, A. Bekesi, P. Lebrun, A.N. Volkov, K. Gevaert, R. Pierattelli, I.C. Felli, P. Tompa. Sci Rep. 2017, 7, 4676
S. Kosol, S. Contreras-Martos, A. Piai, M. Varadi, T. Lazar, A. Bekesi, P. Lebrun, I.C. Felli, R. Pierattelli, P. Tompa Sci. Rep. 2020, 10, 5753.

Key publications from A. Magistrato's Lab

Aupič, J., Lapenta, F., Janoš, P., & Magistrato, A. (2022). Intrinsically disordered ectodomain modulates ion permeation through a metal transporter. Proceedings of the National Academy of Sciences, 119(48), e2214602119.
Spinello, A., Janos, P., Rozza, R., & Magistrato, A. (2023). Cancer-related mutations alter RNA-driven functional cross-talk underlying premature-messenger RNA recognition by splicing factor SF3B. The Journal of Physical Chemistry Letters, 14(27), 6263-6269.
Borišek, J., Saltalamacchia, A., Gallì, A., Palermo, G., Molteni, E., Malcovati, L., & Magistrato, A. (2019). Disclosing the impact of carcinogenic SF3b mutations on pre-mRNA recognition via all-atom simulations. Biomolecules, 9(10), 633.
Aupič, J., Pokorná, P., Ruthstein, S., & Magistrato, A. (2024). Predicting Conformational Ensembles of Intrinsically Disordered Proteins: From Molecular Dynamics to Machine Learning. The Journal of Physical Chemistry Letters, 15(32), 8177-8186.

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Full-length structure of SF3B complex generated using colabfold1.5.2 [M. Milot 2022]. The SF3B complex consists of the SF3B1 (grey), SF3B3 (cyan), SF3B5 (green) and PHF5A (magenta).

Full-length structure of SF3B1 with its ID NTD predicted by the AI-based method Alphafold [J. Jumper 2021]. Colors of the ID NTD account for the level of confidence. This ranges from low (red) to high (blue) level of confidence. Overlap of the structure predicted with Alphafold and a newly released AI-based ESMfold method [Z. Lin 2022].