SPLICING INFO
EURASNET
Leading European laboratories in research on alternative splicing are now united in a European Commission funded Network of Excellence (NoE), EURASNET, the European Alternative Splicing Network. The coordinator of this Network is Reinhard Lührmann, Göttingen. The consortium merges 30 research groups from 25 participating institutions in 13 countries. Over the next five years, this consortium has secured ten million Euros in funding within the Framework 6 Program (FP6) of the European Union, for Research in Alternative Splicing (starting January 01, 2006). The NoE has three important goals:
-
Pursue an ambitious research program via a Joint Research Program.
-
Integrate young investigators in the field into the Network, via a Young Investigator Program.
-
Disseminate awareness of the importance of alternative splicing among medical practitioners, policy makers and the general public.
The joint program of activities (JPA) is organized in 22 work packages covering research, integration and dissemination. The NoE is supported by a scientific advisory board with members J. Dahlberg (University of Madison), W. Filipowicz (Biozentrum Basel), M. Garcia-Blanco (Duke University), A. Krainer (Cold Spring Harbor L), M. Rosbash (Brandeis University), R. Singer (Albert Einstein College) and J. Steitz (Yale University).
Splicing of RNA Molecules
Expression of genes – The way from gene to protein
The genetic information of every
known organism is stored in long chains of DNA (deoxyribonucleic-acid)
molecules. The functional units of the genome are genes, which are arranged in
succession on the DNA strands. Usually one gene codes for one protein, meaning
that the sequence of the DNA determines the sequence of amino acids forming one
specific protein.
However, the information stored in the DNA cannot be translated into a
protein directly, the DNA rather serves as a template which is copied into RNA
(ribonucleic-acid) molecules. This process is called transcription. The
resulting product from transcription, the messenger-RNA (mRNA), is the working
unit of the genome. It provides the link between the message stored in the
genome and the gene product. After transcription the RNA is recognised by a big
cellular machine - called the ribosome – which is able to decipher the
information encoded by the RNA and translates it into a sequence of amino acids,
that forms the protein molecule. This process is called translation.
The succession of events described is called gene expression; it is
performed in the described order in every cell of every living organism known
(Figure 1)
Figure 1:
Schematic overview of the gene-expression pathway in eukaryotic organisms. The
genome is located in the cellular nucleus where it is transcribed and the
pre-mRNA is formed. After several RNA processing steps (including splicing) the
mature mRNA is transported to the cytoplasm where protein production proceeds
(translation).
Removing non-coding sequences: Splicing of pre-mRNA
In the late 1970s electron-microscopic studies of DNA-RNA hybrids showed that mRNA molecules lack stretches of sequences which are present on the chromosomal DNA. When the DNA is transcribed, the primary RNA product shows a sequence that is an exact copy of the DNA template. However, genes and thus also pre-mRNAs are interrupted by stretches of sequences (introns) that are not translated into the amino-acid chain of a protein. In order to produce a functional protein these introns are removed from the primary RNA molecule in a process called splicing: the intervening sequences are cut out and the coding sequences (exons, carrying the genetic information) are fused together (Figure 2).)Figure 2: Splicing of introns in pre-mRNA. The introns are cut out and the exons are fused together. The mature mRNA works as a template for the production of proteins in the next step of gene expression (translation).In eukaryotic organisms pre-mRNAs of protein coding genes are spliced by a huge cellular machinery – the spliceosome. This is a complex of RNA molecules and proteins forming an apparatus that recognises specific sequences and signals on the pre-mRNA defining the start and the end of the intron (Figure 3). The spliceosome catalyses the excision of intronic sequences followed by connection of the exons. After the reaction the intron is released in form of a lariat followed by degradation.
Figure 3:
Sequence elements defining the start and the end of an intron (sequences shown
are typical for vertebrate introns). The 5’ splice site indicates the beginning
the 3’ splice site marks the end of the intron. The branch point nucleotide is
necessary for the splicing reaction (see biochemistry of the splicing reaction
and Figure 4).
Besides spliceosomal splicing there are two additional ways how splicing can
occur, which are referred to later: autocatalytic splicing and enzymatic
splicing. The three possible mechanisms how splicing can occur only differ in
the executing unit, whereas the biochemical reaction is the same.
Biochemistry of the splicing reaction
In the first step of the splicing reaction the branch point nucleotide reacts with the 5’ splice site at the end of the first exon (exon1). Subsequently the last nucleotide of exon1 reacts with the first nucleotide of exon2 and thus forms a continuous nucleotide chain, the intron is released in form of a lariat. The occurring reactions are formally transesterification reactions.
Figure 4: Scheme
of the two transesterification reactions taking place in splicing.
Alternative Splicing – Gaining Diversity and Complexity
In the last decade big efforts have been put into sequencing of the genomes of various organisms, ranging from bacteria and viruses up to the human genome. The knowledge of the exact genetic information shows one remarkable fact: the number of protein-coding genes in higher eukaryotic organisms is much smaller than the number of proteins produced.
Studies on expressed sequences and protein content in human cells suggested that there should be between 100.000 and 150.000 protein-coding genes. However, results from sequencing experiments showed that the human genome only contains around 25.000 genes.
The questions arising from these facts are: What is the reason for this big discrepancy between gene and protein numbers? What is the mechanism that “causes” the complexity of higher eukaryotic organisms even with a relatively low number of genes?
There must be a way to regulate and diversify the function of genes of differentially specialised cell types. Evolution’s solution for the given problem is a mechanism called alternative splicing (AS), which is the most important posttranscriptional regulatory mechanism that causes genome diversity and functional complexity. Alternative splicing means, that during the RNA splicing event, exons can either be retained in the mature message or targeted for removal in different combinations to create a diverse array of mRNAs from a single pre-mRNA. Resulting mature mRNAs are either non-functional or might give rise to proteins with different activities and functions.
About 60% of the human protein-coding genes are alternatively spliced. This explains the fact that the relatively small number of 25.000 genes can lead to a proteome (the total of all proteins in a cell or organism) of several hundred thousands of proteins. Another example is the fruit fly’s (Drosophila melanogaster) genome (14.000 genes) containing about 5.000 genes less than the one of a simple primal nematode (19.000 genes). An outstanding example of an alternatively spliced gene is the so called Dscam gene of the fruit fly. This one gene can be processed into around 38.000 spliced variants.
Alternative splicing patterns
There are several types of alternative splicing events leading to the generation of functionally distinct transcripts (Figure 5):· Use of cassette alternative exons (Figure 5 A)
The most common type of alternative splicing events (one third of all occurring) involves so called cassette type alternative exons. In this case an exon that is flanked by two introns can be spliced out of the transcript together with the introns. Splicing of a cassette exon can result in the complete loss of a certain functional protein domain.
· Use of alternative splice sites (Figure 5 B)
In this case alternative 5’- or 3’- splice sites in exon sequences are chosen. The use of an alternative splice site can lead to subtle changes in the protein activity and therefore leads to a fine tuning of the protein activity.
· Intron retention (Figure 5 C)
Changes in the splicing machinery can lead to a mature mRNA that still contains an intron or parts of intron sequences.
· Mutually exclusion of exons (Figure 5 D)
In this splicing event, an exon can only be included into a mRNA when another exon is spliced out - or vice versa.
· Use of alternative promoters, poly-A sites and terminal introns (Figure 5 E)
Primary RNA transcripts are - additionally to splicing - processed on their 5’- and 3’- ends (5’ capping and 3’ polyadenylation). These modifications are necessary for the protection of mRNAs and also for regulation of translation. Alternative splicing causing an alteration at any of the two RNA ends can thus lead to changes in protein production. On the other hand changes in the 5’ region of the new transcript influences subsequent alternative splicing events further downstream on the same RNA.
Figure 5:
Different variants of alternative splicing events in higher eukaryotes.
Constitutive exons are depicted in dark blue while alternative exons are
represented by bright colouring.
The different alternative splicing events described before can occur everywhere
in the pre-mRNA.
How is everything controlled? - Regulation of alternative splicing
During an alternative splicing event different exons of one transcript are combined in various ways. This process underlies a complex network of regulatory
steps, influenced by a large number of factors and sequences. Splicing enhancers and silencers are sequences on the pre-mRNA which can either lead to the use of
a particular splice site or the skipping of this site. These sequences can be located in exons as well as in introns.
Additionally external signals and factors influence the selection of splice sites. Proteins that bind to the pre-mRNA and / or the spliceosome can influence
the splicing mechanism. Due to the presence of tissue specific splicing factors (proteins) that promote or inhibit splicing, the pre-mRNA is often spliced
different in different types of tissues, depending on the pattern of regulatory factors expressed in these cells. The same principle is used in different
developmental stages of organisms, also providing a different pattern of regulatory factors which control splicing.
Splicing
pathways
Splicing can occur in three different systems, whereas the principal reaction(cutting out introns and fusing the exons) remains the same, but the executive
element differs in the three ways.
Spliceosomal Splicing:
The spliceosome is a huge machine consisting of both proteins and RNA. It can recognise special sequences at the beginning and at the end of each intron (and
in the middle), and removes it from the exonic regions. Afterwards it fuses the exons together. Spliceosomal splicing is the most common splicing mechanism in
eukaryotic organisms.
Autocatalytic Splicing
Autocatalytic splicing basically means that introns can splice themselves out of the mRNA. These RNA molecules are called Ribozymes. The mRNA folds into a
certain structure, which promotes autocatalytic splicing. Autocatalytic introns are not only able to cut out themselves but can also do the reverse reaction of
inserting themselves into RNA or even DNA molecules. Thus they form mobile genetic elements, changing genes and therefore proteins and promoting
evolutional diversity. Autocatalytic splicing is found in eukaryotic organisms (it was discovered in simple eukaryotic organisms), bacteria, mitochondria and
chloroplasts (which are bacteria related organelles) and even in viral RNA molecules.
Enyzmatic Splicing
In the case of enzymatic splicing protein enzymes perform the process of splicing. Ribonucleases cut out the introns and RNA-ligases fuse the exons.
Usually introns in eukaryotic transfer– RNAs (tRNAs) are spliced by this mechanism.