RNA splicing

When splicing or splicing (English splice, connect ',' stick together ') is an important step in the further processing ( processing ) denotes the ribonucleic acid (RNA), which takes place in the nucleus of eukaryotes and in which, from the pre-mRNA to mature mRNA arises.

The initially formed in the transcriptional pre-mRNA contains introns and exons. By splicing the introns are removed and exons linked adjacent to each other to produce mRNA.

Splicing is - together with the capping of the 5 'end and polyadenylation ( tailing ) of the 3' end - during transcription instead, so we speak of a cotranskriptionellen RNA processing and an " RNA factory".

History of discovery

Early genetic studies have shown that gene, mRNA and protein are colinear, which was obvious by the direct transcription or translation. Very nice, this is observed in prokaryotic organisms, where transcription and translation are not separated by compartmentalization of the cell. Even as the RNA polymerase to the DNA, the mRNA synthesized ribosomes can already bind the nascent chain, starting with the translation, which leads to the formation of so-called polysomes.

In eukaryotes, a coupling of transcription and translation is not possible, as a nuclear membrane separates the two processes spatially from one another.

In addition, were able to Chow et al. and Berget et al. the example of adenovirus DNA hybrids show that the mRNA must obviously be subject to an additional processing in eukaryotes, as their internal areas are missing, but which occur in DNA: in very vivid electron microscopic studies of RNA. Could indirectly a maturation by means of the short in comparison to the cytoplasmic RNA half-life of primary transcripts, the so-called heterogeneous nuclear RNA ( hnRNAs ) are shown.

Richard John Roberts and Phillip A. Sharp developed on this basis the concept of split genes and the pre-mRNA splicing, which was rewarded in 1993 with the Nobel Prize for Medicine. Fundamentally new was that the area of a eukaryotic gene on the DNA is repeatedly interrupted by sequences that are not translated into amino acids of the protein later. These so-called intervening sequences, also known as introns, are referred to as a pre-mRNA splicing process from the primary transcript, of the pre-mRNA ( mRNA precursor ), cut out and degraded. Here are simultaneously the two adjacent protein-coding sequence segments, or short exons overexpressed sequences linked together.

A gene can contain up to 60 introns with lengths 35-100000 nucleotides. In addition, the splicing and in mitochondria, archaea and some of the above-mentioned viral RNAs occurs not only in the above-mentioned eukaryotes, but.

Car Catalytic splicing (self - splicing )

Some RNAs may introns without the help of a large spliceosome (see below) to remove. The chemical activity to possess themselves, that is, there are ribozymes that only in some cases ( group II introns) need the help of proteins for proper folding.

1981 were T. Cech et al. evidence for the precursor of the 26S rRNA of Tetrahymena thermophila that for the processing of a 400 -nucleotide-long intron no protein components are required, but that the activity of the RNA itself derives. One speaks therefore of autocatalytic splicing or self- splicing. The discovery of a first ribozyme and thus the catalytic activity of RNA, leading to the postulation of an RNA world in the very early stages of the origin of life, Thomas R. Cech was awarded along with Sidney Altman 1989 with the Nobel Prize for Chemistry. Later studies have shown that self- splicing introns occur in many other organisms. According to the reaction mechanisms of the conserved sequence elements of two RNA species of the self- splicing can be distinguished, the so-called Group I and group II introns. Although it has been shown conclusively that the RNA has the catalytic activity, appear to be involved in the in vivo reactions of the two groups of introns in addition proteins, which probably facilitate the formation of the active structure of the RNA. Since in the described in the following reactions the total number of phosphodiester bonds remains the same because it is transesterification, no energy-supplying cofactors are necessary.

Group I introns occur in the pre- rRNA simple eukaryotes, such as the already mentioned ciliates T. thermophila, and in some pre-mRNAs of cell organelles such as mitochondria and chloroplasts. Excision of the intron takes place in a two step mechanism, as an essential cofactor for the reaction, guanosine, which is brought about by the structure of the RNA in the appropriate position, it then performs a nucleophilic attack on the 5 ' splice site. The nucleofugic group this reaction, the 3'- hydroxy group of the 5 ' exon located now engages in turn as the nucleophile, the 3' splice site at, whereby the two exons with release of the intron linked. In subsequent reactions, the intron eventually closes to form a ring. Let Stereochemical studies of chiral substrates suggest that a single catalytic center catalyzes here both partial reactions of splicing in the form of a round-trip response.

Group II introns are found, however, in pre-mRNAs of mitochondria of yeast and other fungi. A guanosine cofactor is not necessary here, but a 7 or 8 nucleotides of the 3 ' splice site located adenosine in the position to the 5' is the structure of the RNA upstream splice -site nucleophile with its 2 ' hydroxyl group to attack can. This leads to the formation of a unusual 2 ' 5' phosphodiester bond and thus to form a lasso -like structure of the intron, the so-called Lariat. In a second reaction, similar to that of the Group I introns, and finally reaches the 5'- splice site at the 3 ' splice -site nucleophile, resulting in the linking of the two exons and the release of the intron.

When spliceosomal processing of mRNAs (see below ) can be found a the the group II introns of identical reaction mechanism, which has led to a series of speculations that both processes have emerged evolutionarily apart ( see below in the " intron -early " hypothesis ), for example, by fragmentation of a group II introns, or whether it is due to convergent developments by the catalytic optimization of the same chemical reaction.

Splicing of tRNAs

The enzymatic splicing of tRNAs is found in both archaea and eukaryotes, bacteria in contrast, introns in tRNAs are processed by an autocatalytic mechanism that was described in the previous section. The introns in the coding for the tRNA genes are usually found in the anticodon loop directly 3 ' of the anticodon - rare in the Dihydrouracilschleife - and have a length of 14 to 60 bases. When enzymatic splicing, they are in contrast to the splicing of pre-mRNAs do not have their sequence but on a higher-level structure of the whole molecule is detected ( for example, the bulge - helix - bulge motif - BHB motif - in Archaea) and removed in three steps. In this case, the pre- tRNA is first cut twice by an endonuclease that liberates the intron, and two so-called tRNA half-molecules. The resulting cyclic 2 ' 3' phosphate of the 5 ' half of the molecule is then added to a 2 ' ' hydrolyzed OH group, while the 5' phosphate and a 3 OH group of the 3 ' half- molecule is phosphorylated GTP consumption. This allows a ligation of an RNA ligase with ATP hydrolysis. Finally, in the last step, the 2 ' phosphate is removed, which, unusually, vonstattengeht NAD consumption and release of nicotinamide. Also, some mRNAs are processed by a similar, actually very unusual for them mechanism of two endonucleolytic cleavages followed by ligation by tRNA ligase.

Splicing in the spliceosome

The splicing takes place in most cases in a large complex of RNA and proteins place the so-called spliceosome, which catalyzes the reaction in two successive Transesterifikationen. The majority of the intron is removed in this manner. The total number of bonds remains the same for the reaction, energy is only required for the construction and relocation of machinery for catalysis ( spliceosome ). The two individual reactions chemically differ from each other is not, only the positions of the groups involved in the pre-mRNA differ. In both reactions, a nucleophilic substitution (SN1 ) in a phosphate takes place, the nucleophile is one hydroxy group of a ribose.

In the first step attacks the so-called "branch point adenosine " ( located in the middle of the polypyrimidine tract) with the 2' -OH group of the ribose 5 ' splice site that the release of the 5' exons and circularization of introns leads (due to the lasso -like structure " Lariat " called ). In the second step, the now liberated 3'- OH group of the 5'- to the 3'- exon engages splice site, resulting in the linking of the two exons and to release the intron lariat.

The splicing pattern may differ because of the type of tissue and environmental influences. One speaks of alternative splicing, an important basis for a large diversity of proteins. The splicing takes place cotranskriptionell, which means that introns are already removed, even during the polymerase reads the gene.

Other important processes that are occurring during the maturation of pre-mRNA to the mRNA, the

• Capping: Modification of the 5'- end of the RNA with a 7- methylguanosine to improve stability of the RNA and important for translation at the ribosome.
• Tailing: After reaching the Genendes the RNA is approximately 15 nucleotides after a particular base sequence ( AAUAAA ) cut and provided with an approximately 150-200 nucleotide-long poly -A tail. Here, too, a large number of proteins play a role ( CPSF complex CstF complex CFI CFII, PABP2, PAP, etc.), which bind adjacent to the other elements mentioned A2UA3 sequence of the RNA and control the processing of. A termination of transcription - an unfortunately very little understood process in eukaryotes - takes place a little later downstream of the polyadenylation site, including through the TREX complex.

Finally, the mature mRNA through nuclear pores (nuclear pore complex, NPC) is exported from the nucleus to the cytosol, where it is subsequently used in the course of translation to synthesize proteins.

Splicing and disease

Also, for some diseases splicing plays a major role. Mutations in introns have no direct effect on the sequence of the protein encoded by a gene. In some cases, mutations affect sequences important for splicing and thus lead to an incorrect processing of the pre-mRNA. The resulting RNAs encoding non-functional or even harmful proteins and thus lead to hereditary diseases.

A classic example, some forms of beta -thalassemia - an inherited anemia - in which a point mutation that alters the 5 ' splice site of intron 1 of the gene and thus makes it unusable. This means that nearby are detected " cryptic " splice sites and the spliceosome shortened or lengthened generates mRNAs which are translated into inactive proteins. Another well-studied mutation in intron 2 of the same gene leads to the maintenance of a short intron sequence in the final mRNA. In both cases there is a greatly reduced hemoglobin synthesis in erythrocytes and thus resulting in a limited oxygen transport to the clinical picture.

Other cases are, for example, the Ehlers -Danlos syndrome ( EDS) type II ( mutation of a branch -points in the gene COL5A1 ) and spinal muscular atrophy ( a mutation in the gene SMN1 Splicing-Enhancers/Silencers ).

The " RNA factory" (RNA -factory )

In recent years it has become increasingly clear that transcription, processing of RNA (ie, splicing, capping and tailing ), RNA export to the cytoplasm, RNA localization, translation, and RNA degradation influence and regulate each other. The processing of pre-mRNA is still taking place during transcription - Speak of the cotranskriptionellen RNA processing of - and the different machineries take this liaise. For this reason, recently, the term " RNA factory " was coined (RNA -factory ), which should illustrate this. The splicing may also self- refer to the influence processes that occur spatially separated in the cytoplasm. A protein complex which is deposited by the spliceosome on the final mRNA ( exon junction Complex EJC ) enables effective export from the nucleus, transmitting additional information, which allows a subsequent quality control of RNA during translation ( nonsense -mediated mRNA decay [ NMD ] ). Another implication that arises from this is: a full pre-mRNA ( as shown in the illustration above) comes in the living cell actually not before, because introns are removed as just described during transcription.

Splicing and evolution

Many exons encode a functional part of a protein that folds autonomously, a so-called domain. This is the basis for the theory that a modular structure of a gene from exons encoding such protein domains, the opportunity brings with it, use a once evolutionary " invented " domain versatile by combining with others. Thus, by simple recombination of exons according to a modular principle a large variety of protein with diverse functions and properties are created, which is called exon shuffling. A classic example of this is the gene for the protein fibronectin, to the one in the cell adhesion, on the other, with cell migration, proliferation and differentiation is involved. The protein consists mainly of repeats of three protein domains (type III) can be found in addition, also in the plasminogen activator protein (type I) in the blood clotting proteins (type II ), cell surface receptors and proteins of the extracellular matrix.

In addition, there are suspicions that introns already in the last common universal ancestor (last universal common ancestor, an organism from which the three kingdoms Bacteria, archaea and eukaryotes have developed ) may have been already available. This intron -early hypothesis is supported by the discovery of different introns in the genomes of mitochondria, archaea and viruses. Bacteria would have lost their introns according to this theory, which could be explained by an optimization of the genome for rapid proliferation, and short generation time. In contrast, at least some of the introns of this theory do not seem to correspond, as they have presumably developed from other precursor sequences. Thus, there may have been no " Ur - intron " ( from which all present-day introns have emerged ), but rather more as an ancestor sequences for the introns known today. Thus, introns were not monophyletic, but would most closely match a polyphyletic group. This relationship is expressed in the intron -late hypothesis.