Discovery of how a key enzyme of the spliceosome exerts its controlling function
DNA (deoxyribonucleic acid) is the carrier of genetic information in all living organisms. Certain regions of DNA, called genes, contain the information that is needed for the assembly of proteins – the molecules that are responsible for most cellular functions. In humans, and in other higher organisms, most genes are built in a mosaic-like fashion – sections that encode the design plans of proteins alternate with so-called non‑coding sections. To produce a protein from the gene that encodes it, first a copy of the gene in the form of RNA (ribonucleic acid, a relation of DNA) has to be produced. From this RNA molecule, the non‑coding sections are removed and the coding sections are spliced together. The result is the so-called “messenger RNA”, the mature RNA that directs protein synthesis. This essential RNA maturation process is termed “RNA splicing”.
The process of splicing is carried out by a highly complex molecular machine termed the spliceosome. Human spliceosomes are built up from protein and RNA molecules. They contain some 170 different proteins and five RNA molecules termed “small nuclear RNAs” (snRNAs). It is currently believed that certain snRNAs represent the tools with which the spliceosome carries out the cutting and joining of RNA sections, turning the messenger RNA's precursor (“pre‑mRNA”) into mature messenger RNA. The proteins of the spliceosome are needed to bring these tools to the right place at the right time, and to set them into operation. Splicing processes in higher organisms are very highly regulated. In fact, differing patterns of excision and joining of a given pre‑mRNA molecule can lead to any one of a selection of different mature mRNA molecules – all from the same gene. This ability to select the mRNA product according to need is termed “alternative splicing”, and it is thought to be the most important means by which human cells manage to produce a vast spectrum of different proteins from a relatively restricted number of protein-encoding genes.
For every splicing step (removal of a single piece of non-coding RNA), a new spliceosome is assembled on the precursor mRNA molecule. The cutting of the pre‑RNA only takes place once the target splicing site has been identified. The cutting tools of the spliceosome's snRNA are brought into position, but first in an inactive form; they are packed into other components of the spliceosome, rather like a knife that is (initially) kept safely in its sheath. On receiving a particular “start signal”, the molecular knife is drawn and put to use. Until recently, it was only known that a certain protein (known as Brr2) was responsible for activating this molecular knife. Brr2 belongs to a family of enzymes that are called “RNA helicases” due to their ability to separate RNA molecules that are paired with one another (by unwinding the RNA helices, hence the name). In this way Brr2 sets free an snRNA “knife” and allows it to do its job. However, Brr2 also possesses a remarkable molecular architecture, which distinguishes it from other helicases (for details, see “Further publications” no. 1). Until now it was not known how this special architecture is put to use in the cell to regulate the function of Brr2.
Now researchers at the Max Planck Institute for Biophysical Chemistry in Göttingen and at the Freie Universität Berlin have jointly discoverd the mechanism by which this regulation takes place. Sina Mozaffari-Jovin and Cindy Will, from the research group of Reinhard Lührmann in Göttingen, discovered by means of biochemical studies that the helicase activity of Brr2 is inhibited by a particular part of another protein of the spliceosome, Prp8. “Brr2 is, so to speak, held on a short leash by Prp8, preventing it from setting the cutting tools of the spliceosome into action,” explains Reinhard Lührmann.“This prevention requires direct contact between the Prp8 molecule and the helicase Brr2.” Following up on this work Traudy Wandersleben and Karine Santos, from the research group of Markus Wahl in Berlin, determined the atomic structure of the Brr2 protein in contact with the relevant regulatory portion of Prp8. “To do this we used X‑ray crystallography,” states Markus Wahl. “There are excellent facilities for this kind of research at the BESSY II synchrotrons at the Helmholtz Centre in Berlin, where the necessary specialized instrumentation is available”. The atomic structure explains the biochemical observations in a clear and elegant way: At the end of the Prp8 molecule there is an elongated region of protein that blocks a central channel of the Brr2 helicase and, by doing so, prevents the RNA molecules that are separated by Brr2 from binding to the helicase.