FAQ

What is spliceosome?

The spliceosome (SPL) is a large molecular machine found in the nucleus of eukaryotic cells. It is responsible for removing non-coding sequences called introns from pre-mRNA, leaving behind only the protein-coding sequences called exons. This process, called splicing, is essential for the production of mature mRNA, which is then translated into proteins. Splicing is a complex process that involves many different proteins and RNA molecules. The SPL consists of 5 small nuclear (sn)RNAs and over 100 proteins.

The SPL recognizes specific sequences at the beginning and end of introns, and then cuts out the introns and splices together the exons. This process is highly regulated, and errors in splicing can lead to the production of defective proteins, which can cause a variety of diseases.

Do spliceosome contains IDPs/IDRs?

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 [Korneta 2012]. 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 [Cretu 2016].

What is intrinsically disordered protein and why these are important?

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) are proteins that don't have a fixed 3D structure. This lack of structure allows them to be very flexible and adaptable, which is important for many cellular functions. For example, IDPs/IDRs can bind to a variety of different partners, which allows them to play a role in signaling pathways, cell cycle regulation, and other important processes. IDPs/IDRs are also important for protein folding and assembly. Some IDPs act as molecular chaperones, helping other proteins to fold into their correct shape.  IDPs/IDRs undergoes different Post-translational modifications (PTMs) which can further increase their flexibility and ability to interact with other molecules.

What are the current state of art for studying IDPs/IDRs?

The state of the art for studying IDPs/IDRs experimentally is constantly evolving. As a result, the state of the art for studying IDPs/IDRs is likely to change in the future.

Here are some of the most common techniques used: 

Nuclear Magnetic Resonance (NMR) spectroscopy: NMR is a powerful technique for studying the structure and dynamics of IDPs/IDRs. It can provide information about the secondary structure, flexibility, and interactions of these proteins. 

Circular Dichroism (CD) spectroscopy: CD can be used to study the secondary structure of IDPs/IDRs. It is a relatively simple and fast technique that can be used to monitor changes in protein structure upon binding to other molecules. 

Small-Angle X-ray Scattering (SAXS): SAXS can be used to study the overall shape and size of IDPs/IDRs. It can also be used to study the interactions of IDPs/IDRs with other molecules. 

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): HDX-MS can be used to study the dynamics of IDPs/IDRs. It can provide information about the regions of the protein that are flexible and accessible to solvent. 

Single-Molecule Fluorescence Spectroscopy (SMFS): SMFS can be used to study the dynamics of individual IDP/IDR molecules. It can provide information about the folding and unfolding of these proteins. 

Computational methods: Computational methods, such as molecular dynamics (MD) simulations and coarse-grained (CG) modeling, can be used to study the structure and dynamics of IDPs/IDRs. These methods can complement experimental data and provide insights into the mechanisms of IDP/IDR function. 

The choice of technique depends on the specific questions being asked and the properties of the IDP/IDR being studied. For example, NMR is well-suited for studying the structure and dynamics of small IDPs/IDRs, while SAXS is better suited for studying the overall shape and size of larger IDPs/IDRs.

Do force fields are reliable for studying IDPs/IDRs using MD simulation?

From last several decades we have well optimised force fields (FFs) for foleded proteins, but for IDPs/IDRs these are not much reliable [1, 2]. In this direction there are several groups attempting to find suitable FF and water models which can predict reliable ensamble for IDPs/IDRs. currently AMBER-disp, CHARMM36m, AMBER99SB-ILDN with combination with TIP4PD/AMBER-disp water models are better for simulating IDPs/IDRs [3-6]. Modified CG FFs (SIRAH2.2/MARTINI3) are also now days used for IDPs/IDRs simulation [7-11].

References:

1. Zapletal, Vojtěch, et al. "Choice of force field for proteins containing structured and intrinsically disordered regions." Biophysical journal 118.7 (2020): 1621-1633.

2. 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.

3. Piana, S., Donchev, A. G., Robustelli, P., & Shaw, D. E. (2015). Water dispersion interactions strongly influence simulated structural properties of disordered protein states. The journal of physical chemistry B, 119(16), 5113-5123.

4. Lindorff‐Larsen, K., Piana, S., Palmo, K., Maragakis, P., Klepeis, J. L., Dror, R. O., & Shaw, D. E. (2010). Improved side‐chain torsion potentials for the Amber ff99SB protein force field. Proteins: Structure, Function, and Bioinformatics, 78(8), 1950-1958.

5. Huang, J., Rauscher, S., Nawrocki, G., Ran, T., Feig, M., De Groot, B. L., ... & MacKerell Jr, A. D. (2017). CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nature methods, 14(1), 71-73.

6. Robustelli, P., Piana, S., & Shaw, D. E. (2018). Developing a molecular dynamics force field for both folded and disordered protein states. Proceedings of the National Academy of Sciences, 115(21), E4758-E4766.

7. Ramis, R., Ortega-Castro, J., Casasnovas, R., Mariño, L., Vilanova, B., Adrover, M., & Frau, J. (2019). A coarse-grained molecular dynamics approach to the study of the intrinsically disordered protein α-synuclein. Journal of chemical information and modeling, 59(4), 1458-1471.

8. Klein, F., Barrera, E. E., & Pantano, S. (2021). Assessing SIRAH’s capability to simulate intrinsically disordered proteins and peptides. Journal of chemical theory and computation, 17(2), 599-604.

9. Thomasen, F. E., Pesce, F., Roesgaard, M. A., Tesei, G., & Lindorff-Larsen, K. (2022). Improving Martini 3 for disordered and multidomain proteins. Journal of Chemical Theory and Computation, 18(4), 2033-2041.

10. Thomasen, F. E., Skaalum, T., Kumar, A., Srinivasan, S., Vanni, S., & Lindorff-Larsen, K. (2024). Rescaling protein-protein interactions improves Martini 3 for flexible proteins in solution. Nature Communications, 15(1), 6645.

11. Souza, P. C., Alessandri, R., Barnoud, J., Thallmair, S., Faustino, I., Grünewald, F., ... & Marrink, S. J. (2021). Martini 3: a general purpose force field for coarse-grained molecular dynamics. Nature methods, 18(4), 382-388.