The spliceosome: NMR spFRET dice up protein complex

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  • Published: Dec 1, 2016
  • Author: David Bradley
  • Channels: NMR Knowledge Base
thumbnail image: The spliceosome: NMR spFRET dice up protein complex

Function and form

Slicing the spliceosome: NMR dices protein complex The large subunit of U2AF binds to the mRNA precursor. Picture: Christoph Hohmann, NIM

Prior to gene expression in the cell, the non-coding regions of the genome have to be removed by the spliceosome. A joint single pair Förster resonance energy transfer (spFRET) and nuclear magnetic resonance (NMR) spectroscopy study by researchers in Munich suggests that distinct conformations of a member of this molecular complex play a vital role in the process, with implications for biomedical science.

Cells convert DNA to RNA to template protein production but before that final step, gene expression, the non-coding regions of the genome have to be removed by the so-called spliceosome. A nuclear magnetic resonance (NMR) spectroscopy study by researchers in Munich suggests that distinct conformations of a member of this molecular complex play a vital role in the process, with implications for biomedical science.

Ribonucleic acid, RNA, is the intermediate between stored genetic information and the protein products synthesised using the genetic templates. Within the cell nucleus, defined segments of the DNA are first transcribed into RNA copies called messenger RNA precursors (pre-mRNAs). In many cases, these primary transcripts contain interspersed sequences that interrupt the actual protein-coding sequence. These "introns" must be removed and the coding sequences spliced before the information can be used for protein synthesis. Moreover, given that a single gene may actually encode for several different forms of a protein by a process called alternative splicing, the differently spliced mRNA strands play a crucial role in post-transcriptional gene regulation. Different splices for different protein forms can have different functions, in other words.

Splice and dice

All of the splicing operations in the cell are undertaken by a molecular machine known as the spliceosome, a term coined analogously to the ribosome, the "some" meaning body. Now, Don Lamb of the Department of Chemistry at LMU (Ludwig- Maximilians-Universität München) and Michael Sattler of the Helmholtz Zentrum München and the Technical University of Munich (TUM), have successfully demonstrated that the distinct structural configurations adopted by a protein which is essential for assembly of the spliceosome on mRNA precursors have an important effect on splicing efficiency. The team describes details of their findings in the journal Proceedings of the National Academy of Sciences.

The human spliceosome has several subunits that have to be assembled on to the mRNA precursor through a choreographed sequence of steps. The way in which the individual subunits bind affects the assembly and function of the molecular machine. "The assembly factor we have studied, called the U2 Auxiliary Factor or U2AF for short, is critical for the correct recognition of the splicing sites at one end of the introns," explains team member and first author on the PNAS paper, Lena Voithenberg. U2AF itself is made up of two different subunits but in its free form, the larger of the two subunits is a highly dynamic protein, a characteristic demonstrated by Voithenberg and her colleagues using spFRET. NMR spectroscopy was carried out in parallel at the Bavarian NMR Center (run jointly by the Helmholtz Zentrum München and the TUM), which provided additional information relating to the structure and conformational dynamics of U2AF.

Changing expression

"We found that the large subunit rapidly switches its conformation from an open to a closed structure on timescales of micro- to milli-seconds," explains Voithenberg. Importantly, it is only the open form that can actually bind to the RNA. Moreover, the proportion of molecules in this conformation depends on the relative binding affinity of the RNA sequences available. If the sequences have a high affinity for the binding subunit then they will have a higher probability of being recognized - and so cleaved - than those that have a lower affinity.

The researchers suggest that their discovery of different structural conformations adopted by the large subunit of U2AF and the equilibrium between the different conformations regulates the splicing efficiency at different splicing sites. It is this phenomenon that has implications for how mRNA precursors are cleaved and spliced, which in turn affects not only the structure of the final protein, but also the rate at which it is expressed in the cell. Appropriate and efficient gene expression represents a healthy cell but when the system goes awry disease can arise. Thus, understanding the way in which the spliceosome functions and putatively malfunctions could be critical to understanding diseases involving errant gene expression.

"We will next look into modified RNAs and how they influence the conformational dynamics of U2AF as well as the effect of additional binding partners," Lamb told SpectroscopyNOW.

Related Links

Proc Natl Acad Sci 2016, online: "Recognition of the 3' splice site RNA by the U2AF heterodimer involves a dynamic population shift"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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