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Reinhard Lührmann
Director

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Juliane Moses
Assistant
Phone:+49 551 201-1405Fax:+49 551 201-1197
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Berthold Kastner
Coordinator
Phone:+49 551 201-1313Fax:+49 551 201-1197

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Yearbook Articles (in German)

Reinhard Lührmann

Cellular Biochemistry

The production of proteins in the cells of higher organisms is a complex process involving many steps. First, the genetic information for a protein is re written from DNA into a working copy, the precursor messenger RNA (pre mRNA). However, pre-mRNAs contain regions that do not contain information used for the production of proteins (the so-called “introns”). These regions must be precisely cut out and the remaining regions, which contain usable information (the “exons”), linked together. This maturation process is termed "pre-mRNA splicing”. Only mature mRNAs, that are transported from the cell nucleus into the cytoplasm, can be used by the ribosome as a template for the production of proteins.


The presence of exons and introns is a great advantage for an organism, as different combinations of exons from a given pre-mRNA species can be chosen to be included in the mature mRNA product. In this way, mRNAs corresponding to many different proteins can be made from a single gene. This so-called alternative splicing represents an additional level at which gene expression can be regulated, and leads to an enormous increase in the genetic capacity of higher eucaryotes. This explains why humans manage with only just over 20,000 protein-encoding genes in their genomes. Understanding splicing at the molecular level is of great medical relevance, as aberrant pre-mRNA splicing is the basis or a severity modifier of a plethora of human diseases.


The pre-mRNA splicing reaction takes place in two steps. Both involve phosphoester-transfer reactions, and both are carried out by a macromolecular machine, the spliceosome. Spliceosomes consist of well over 100 proteins and five small RNA molecules (the snRNAs U1, U2, U4, U5 and U6) and thus consist largely of protein. Many of the spliceosome's components are organised into smaller, stable sub-complexes. For example, about 50 of the spliceosomal proteins are stably bound to the snRNAs, forming RNA–protein particles (termed small nuclear ribonucleoproteins or snRNPs) which include the U1 and U2 snRNPs and the U4/U6.U5 tri-snRNP.

<p>Assembly of the spliceosome by the stepwise binding of the snRNPs to the pre-mRNA. In the early phase of spliceosome assembly, the U1 snRNP binds to the 5' splice site (5' SS: where exon 1 ends and the intron begins), and the U2 snRNP binds to the so-called branch point (BP: near the 3' end of the intron). This spliceosome assembly intermediate is called the A complex. The subsequent binding of the U4/U6.U5 tri snRNP complex gives rise to the precatalytic B complex. The catalytic activation of the spliceosome takes place in two steps. In the first, the RNA helicase Brr2 acts to produce the B<sup>act</sup> complex and in the second, the RNA helicase Prp2 facilitates the formation of the B* complex. This has a functional active site and, following the recruitment of the protein Cwc25, the first step of splicing takes place. In this step, the phosphodiester bond at the 5' splice site is cleaved and, at the same time, the 5' end of the intron becomes linked to the 2' hydroxyl group of an adenosine at the branch point. In the next step, the RNA helicase Prp16 converts the spliceosome to the C* complex, which – with the help of the proteins Prp18 and Slu7 – carries out the second catalytic step of the splicing reaction. In this step, the phosphodiester bond at the 3' splice site (3' SS: where the intron ends and exon 2 begins) is cleaved and at the same time the two exons are joined to one another. The intron is released from the spliceosomal complex in the form of a lasso (lariat) and the snRNPs are recycled for subsequent rounds of splicing.  The dissociation phase of the spliceosome requires catalysis by the RNA helicases Prp22 and 43.  "ATP" indicates the steps that require ATP molecules as a source of chemical energy.<span size="2" color="black" face="Calibri" style="color: black; font-family: Calibri; font-size: x-small;"><br /></span></p> Zoom Image

Assembly of the spliceosome by the stepwise binding of the snRNPs to the pre-mRNA. In the early phase of spliceosome assembly, the U1 snRNP binds to the 5' splice site (5' SS: where exon 1 ends and the intron begins), and the U2 snRNP binds to the so-called branch point (BP: near the 3' end of the intron). This spliceosome assembly intermediate is called the A complex. The subsequent binding of the U4/U6.U5 tri snRNP complex gives rise to the precatalytic B complex. The catalytic activation of the spliceosome takes place in two steps. In the first, the RNA helicase Brr2 acts to produce the Bact complex and in the second, the RNA helicase Prp2 facilitates the formation of the B* complex. This has a functional active site and, following the recruitment of the protein Cwc25, the first step of splicing takes place. In this step, the phosphodiester bond at the 5' splice site is cleaved and, at the same time, the 5' end of the intron becomes linked to the 2' hydroxyl group of an adenosine at the branch point. In the next step, the RNA helicase Prp16 converts the spliceosome to the C* complex, which – with the help of the proteins Prp18 and Slu7 – carries out the second catalytic step of the splicing reaction. In this step, the phosphodiester bond at the 3' splice site (3' SS: where the intron ends and exon 2 begins) is cleaved and at the same time the two exons are joined to one another. The intron is released from the spliceosomal complex in the form of a lasso (lariat) and the snRNPs are recycled for subsequent rounds of splicing.  The dissociation phase of the spliceosome requires catalysis by the RNA helicases Prp22 and 43.  "ATP" indicates the steps that require ATP molecules as a source of chemical energy.

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Spliceosomes do not exist in the cell nucleus as complete, pre-formed complexes. Rather, a new spliceosome is built up from its components around each intron that requires excision (Figure 1). First, the U1 and U2 snRNPs recognize and bind the 5'ss and of the pre-mRNA. The resulting complex is termed the A complex. Subsequent binding of the U4/U6.U5 tri snRNP leads to the formation of the so-called B complex. However, this multi-megadalton complex still has no catalytically active site. The subsequent catalytic activation of the spliceosome involves dramatic structural rearrangements that lead to changes in the conformations of its snRNAs and also its biochemical composition. During this process, a complex network of RNA–RNA interactions is formed between the pre mRNA and the snRNAs U2, U5 and U6. This network forms the heart of the spliceosome's catalytic center (Figure 2). The catalytically activated spliceosome is now ready to perform the first step of the splicing reaction. The product of this first step is the C complex, which then catalyses the second step. After this, the excised intron and remaining snRNPs are separated from the mature (spliced) mRNA, and the snRNPs are actively released to take part in a new round of splicing.

<p>Figure 2:  Dynamics of the spliceosomal RNA–RNA network. The figure shows the most important RNA base-pairing interactions as "ladders" in the pre-catalytic complex B (on the left) and in the activated spliceosome (on the right). To activate the spliceosome, the base-pairing between the U4 and U6 snRNAs, and also between the U1 snRNA and the 5' splice site, is disrupted. At the same time the U6 snRNA enters into new base-pairing interactions with the 5' splice site and the U2 snRNA. This RNA network (yellow background) provides the core of the spliceosome's catalytic center. The positioning of the 5' splice site in exon 1 is further supported by interactions between the U5 snRNA and the 3' end of exon 1. The snRNAs are shown schematically according to their known two-dimensional folding. The intron of the pre mRNA is shown as a thin purple line.<span size="2" color="black" face="Calibri" style="color: black; font-family: Calibri; font-size: x-small;"><br /></span></p> Zoom Image

Figure 2:  Dynamics of the spliceosomal RNA–RNA network. The figure shows the most important RNA base-pairing interactions as "ladders" in the pre-catalytic complex B (on the left) and in the activated spliceosome (on the right). To activate the spliceosome, the base-pairing between the U4 and U6 snRNAs, and also between the U1 snRNA and the 5' splice site, is disrupted. At the same time the U6 snRNA enters into new base-pairing interactions with the 5' splice site and the U2 snRNA. This RNA network (yellow background) provides the core of the spliceosome's catalytic center. The positioning of the 5' splice site in exon 1 is further supported by interactions between the U5 snRNA and the 3' end of exon 1. The snRNAs are shown schematically according to their known two-dimensional folding. The intron of the pre mRNA is shown as a thin purple line.

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Both the snRNAs and the spliceosomal proteins are essential for the function of the spliceosome. They are involved in the recognition of the pre-mRNA's splice sites and in the formation of the spliceosome's catalytic center. Furthermore, a number of energy-requiring enzymes – the so-called RNA helicases – play decisive roles in the stepwise structural rearrangements of the spliceosome (Figure 1).


The primary goal of our research is to understand the structure and the function of the splicing machinery. One main question that we wish to address is how the structural rearrangements of the spliceosome during its work cycle are directed and regulated. Another is what is the nature of the catalytic center of the spliceosome – for example, does it consist only of RNA components (like a ribozyme), or do RNA and protein both contribute to catalysis (as in an RNP enzyme)? To answer these questions we are using an integrated experimental approach that involves a broad palette of methods. We are using biochemical and molecular-genetic methods to study the functions of the proteins and snRNA molecules in splicing, mainly by focussing on the spliceosomes of human cells and those of baker's yeast. At the same time we are using electron cryomicroscopy, X-ray crystallography, mass spectrometry, and fluorescence spectroscopy to investigate the spatial organization and the structural dynamics of isolated spliceosomes.


 
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