How was alternative splicing discovered

Structure and function of spliceosomes

Eukaryotic pre-mRNA contains non-coding regions (introns) which have to be removed before the mRNA can be used in protein synthesis. This so-called splicing is catalyzed in the cell nucleus by the spliceosome, a molecular machine of high complexity and dynamics that consists of numerous protein and RNA components and is newly formed on each intron to be removed. The structure and function of the catalytic working cycle of the spliceosome are examined using biochemical, molecular biological, genetic and structural biological methods.

The formation of proteins in cells of higher living beings is a complex, multi-stage process. First, the genetic information for a protein is transcribed from the DNA into a working copy, the precursor messenger RNA (pre-mRNA). However, these RNA copies can only be used for protein production if the internal areas, so-called “introns”, which are not required, have been removed through precise cutting and the information-relevant areas, “exons”, have been re-linked. This maturation process is known as mRNA splicing. Only this mature mRNA can be transported from the cell nucleus into the cytoplasm and used by the ribosomes as a template for the formation of proteins. This exon-intron arrangement offers the organism great advantages: by alternative splicing, i.e. by optionally linking different exons, mRNAs for different proteins can be produced from one gene. Alternative splicing thus represents an additional level of regulation of gene expression, which enormously increases the genetic capacity of higher eukaryotes. This also explains why humans can get by with just over 20,000 protein-coding genes in their genome.

The mRNA splicing reaction takes place in two steps via phosphoester transfer reactions and is brought about in the cell nucleus by the molecular machine of the spliceosome. Spliceosomes are made up of well over 100 proteins and five small RNA molecules (the snRNAs U1, U2, U4, U5 and U6) and are therefore gigantic, very protein-rich nanomachines. Many of these building blocks are organized in stable sub-complexes. So store z. B. approx. 50 spliceosomal proteins with the snRNAs to form RNA protein particles, the snRNPs (small nuclear ribonucleoproteins) U1, U2 and the U4 / U6.U5-tri-snRNP.

Spliceosomes do not exist in the cell nucleus as prefabricated machines, but are built anew from their building blocks on each intron to be spliced ​​(Fig 1). First, the snRNPs U1 and U2, together with some helper proteins, recognize and bind the beginning and end of an intron. The binding of the U4 / U6.U5-tri-snRNP then leads to the formation of the so-called B complex. However, this multi-mega-dalton complex does not yet have an active catalytic center. Gradual dramatic structural changes of the B-complex spliceosome alter the conformations of the snRNAs and the original biochemical composition of the B-complex. A complex network of RNA-RNA interactions forms between the pre-mRNA and the snRNAs U2, U5 and U6, which is to be regarded as the heart of the catalytic center (Fig. 2). The catalytically activated spliceosome is now able to complete the first step of the splicing reaction, with the formation of the C-complex spliceosome. After the second catalytic step has taken place, the spliceosome together with the excised intron is detached from the mature mRNA and dissociated into its individual parts. Both snRNAs and spliceosomal proteins are essential for the function of the spliceosome. Among other things, they are involved in the detection of the splice points and the formation of the catalytic center. Furthermore, a number of energy-consuming enzymes, so-called RNA helicases, play a decisive role in the gradual rearrangements of the spliceosome (Fig. 1).

One of the main goals of Reinhard Lührmann's researchers is to understand how the splicing machinery works and how it is structured. On the one hand, the question of how the structural changes of the spliceosome take place during its working cycle and how they are regulated is in the foreground. On the other hand, the scientists want to clarify the question of how the catalytic center of the spliceosome is structured, i.e. H. like a ribozyme, does it only consist of RNA components or does it act more like an RNP enzyme in which RNA and proteins contribute equally to catalysis? In order to be able to answer these questions, they use an integrated experimental approach that encompasses a broad methodological spectrum. They analyze the function of proteins and snRNA molecules during splicing using biochemical and molecular genetic methods, with the researchers primarily concentrating on the investigation of spliceosomes from human cells and baker's yeast. At the same time, they use cryo-electron microscopy and X-ray crystallography as well as mass spectrometric and fluorescence spectroscopic methods to investigate the spatial structure and structural dynamics of isolated spliceosomes.

Spliceosomes are protein-rich RNP machines with a very dynamic biochemical composition

The biochemical composition of the splicing machines changes during a work cycle, i. H. during the splicing of a single intron, continuously. It is therefore not possible to isolate endogenous biochemically uniform spliceosomes from cells. Therefore, methods had to be developed that make it possible to isolate biochemically uniform functional states of a spliceosome. To do this, a In vitro-transcribed intron-containing model pre-mRNA incubated with cell extracts to allow the snRNPs to attach to the pre-mRNA. Under suitable conditions, spliceosome complexes can be isolated that are locked in a certain functional state. This created the prerequisite for first characterizing the protein composition of the most important functional stages of human spliceosomes using mass spectrometry. This inventory showed that a total of approximately 170 proteins with the in vitro assembled splicing machinery, with each individual functional stage containing significantly fewer proteins. Many of the proteins show various post-translational modifications. The mass spectrometric determination of the phosphopeptides showed that about a third of all spliceosomal proteins are phosphorylated, with some phosphorylations only occurring in very specific functional stages of the spliceosome and acting as molecular switches for structural changes in the spliceosome. For example, the SRPK2 and PRP4 kinases phosphorylate, inter alia. Proteins of the U4 / U6.U5-tri-snRNP particle as a prerequisite for its stable integration into the spliceosome.

Also spliceosomes of the fruit fly Drosophila melanogaster and the baker's yeast S. cerevisiae were isolated and characterized biochemically. The metazoan spliceosomes (Drosophila and human) show a striking match in their protein composition, both in terms of the number and identity of the proteins. The total number of proteins found in the yeast spliceosome, on the other hand, is about 95, significantly lower than that of the metazoan spliceosomes. Many of the metazoan-specific proteins are involved in the regulation of alternative splicing, which hardly plays a role in yeast. For almost every yeast spliceosomal protein there is an evolutionarily conserved homolog in metazoan spliceosomes. One can therefore assume that we are dealing with the evolutionarily conserved core splicing machinery in yeast.

If one compares the protein composition of the functional stages of the spliceosomes, a first insight into the dynamic events of the spliceosomal protein composition emerges (Fig. 3). So leave z. For example, during the transition from the pre-catalytic to an activated spliceosome (see Fig. 1), in addition to the snRNAs U1 and U4, about 35 proteins form the yeast spliceosome, including all U1 and U4 / U6-snRNP- and some U5-specific Proteins, ie the structures of some snRNPs, change drastically during the catalytic activation of the spliceosome. On the other hand, more than 20 proteins (including 12 new) are stably integrated into the activated spliceosome (Bact complex). Several of these proteins are of particular importance in the simultaneous rearrangement of the RNA-RNA network of the spliceosome (Fig. 2). From the proteomic studies it is also clear that the dynamics in the biochemical composition of the spliceosome during its functional cycle is a design principle that has been evolutionarily conserved from yeast to humans (employees: Anokhina, Bessonov, Dannenberg, Dönmez, Fabrizio, Hartmuth, Herold, Mathew, Schneider, Will, in collaboration with AG H. Urlaub; MPI for Biophysical Chemistry).

In vitro-Reconstitution of the catalytic phase of yeast spliceosomes from purified components

Ideally, the role of individual proteins or snRNAs in the splicing mechanism would be investigated in a test tube in a spliceosome completely reconstituted from the individual components. The large number of proteins and snRNPs involved and the dynamics of the assembly process of the spliceosomes make such a system, which only uses recombinantly produced proteins, almost impossible. However, a partial complementation strategy in the yeast system made it possible for the first time to complete the entire catalytic phase with purified components in vitro to reconstitute. With this system, the individual steps of the catalytic activation of the spliceosome could be analyzed and the function of individual proteins during the catalytic phase could be elucidated. This also led to the discovery and functional description of a new protein required for the first catalytic step, Cwc25 (Fig. 1). Using the method of fluorescence correlation spectroscopy, the dynamics of the proteins during the catalytic phase of the spliceosome were recorded in a quantitative and time-resolved manner. Furthermore, the end phase of the splicing cycle, i.e. H. the dissociation of the purified intronlariat spliceosome into its individual parts, in vitro understandable (employees: Fabrizio, Fourmann, Odenwälder, Ohrt, Schmitzova, Warkocki, in collaboration with J. Enderlein, University of Göttingen).

Structure of the catalytic core RNPs of spliceosomes

To answer the question of which proteins are necessary to generate and maintain the catalytically active RNA network of the spliceosome, purified human C-complex spliceosomes were subjected to stringent biochemical conditions in order to identify the proteins that are very tightly bound to the spliceosome stay. In 1 molar sodium chloride solution, a macromolecular spliceosome complex including the RNA network remains intact, which contains approx. 40 of the more than 100 proteins of the native C complex. Using cross-linking strategies, it was possible to identify the proteins that are in direct contact with catalytically important RNA elements and thus lie in the heart of the catalytic center. Chemical modification studies of RNA as well as RNA-RNA cross-links in human spliceosomes provided a detailed picture of the RNA-RNA interaction network in the catalytically active spliceosome. On the basis of these structural data, a first 3D model of the RNA-RNA network in the catalytic center could be created (employees: Anokhina, Bessonov, Fabrizio, Hartmuth, Rasche, Will, in collaboration with the H. Urlaub group (Max-Planck Institute for Biophysical Chemistry) and E. Westhof (IBMC Strasbourg).

Investigations into the 3D structure of isolated snRNPs and spliceosomes