Splicing (biology)
When splicing or splicing ( English splice , connect ',' stick together ') is an important step of further processing ( processing of) ribonucleic designated (RNA) which in the cell nucleus of eukaryotic takes place and in which from the pre-mRNA the mature mRNA arises.
The pre-mRNA initially formed in the transcription still contains introns and exons . Splicing removes the introns and links the adjacent exons to form the finished mRNA.
Splicing takes place together with the polyadenylation (tailing) of the 3 'end after transcription, so it is a post-transcriptional process. In contrast, capping the 5 'end is a co-transcriptional process.
Story of discovery
Early genetic studies could show that gene , mRNA and protein are colinear, which was obvious from direct transcription or translation. This can be observed very well in prokaryotic organisms, where transcription and translation are not separated from one another by compartmentalizing the cell. While the RNA polymerase is still synthesizing the mRNA on the DNA, ribosomes can already bind the nascent chain and begin translation, which leads to the formation of so-called polysomes .
In eukaryotes, however , a coupling of transcription and translation is not possible because a nuclear membrane spatially separates the two processes.
In addition, Chow et al. and Berget et al. in very descriptive electron microscopic examinations of RNA: DNA hybrids using the example of adenoviruses show that the mRNA in eukaryotes must obviously be subject to additional processing, since it lacks internal areas, but these are found in the DNA. Maturation could be shown indirectly based on the short half-life of primary transcripts, the so-called heterogeneous nuclear RNAs (hnRNAs), compared to cytoplasmic RNAs.
On this basis, Richard John Roberts and Phillip A. Sharp developed the concept of split genes and pre-mRNA splicing, which was awarded the 1993 Nobel Prize for Medicine. What was 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 later protein. These so-called intervening sequences , also known as introns , are cut out and degraded from the primary transcript, the pre-mRNA (precursor mRNA), in a process known as pre-mRNA splicing . The two adjacent protein-coding sequence segments, or exons for expressed sequences for short , are linked to one another at the same time .
A gene can contain up to over 60 introns with lengths between 35 and 100,000 nucleotides. In addition, splicing occurs not only in the eukaryotes mentioned, but also in mitochondria , archaea and some of the viral RNAs mentioned above.
Autocatalytic splicing (self-splicing)
Some RNAs can remove introns without the help of a large spliceosome (see below). They themselves have the chemical activity for this, i. H. they are ribozymes which only in some cases (group II introns) require the help of proteins for correct folding.
In 1981 T. Cech et al. to prove for the precursor of the 26S rRNA from Tetrahymena thermophila that no protein components are required for the processing of an intron about 400 nucleotides long , but that the activity comes from the RNA itself. One therefore speaks of autocatalytic splicing or self-splicing . For this discovery of a first ribozyme and thus the catalytic activity of RNA, which led to the postulation of an RNA world in the very early phase of life, Thomas R. Cech was awarded the Nobel Prize in Chemistry in 1989 together with Sidney Altman . Later studies could show that self-splicing introns occur in many other organisms. According to the reaction mechanisms and the conserved sequence elements of the RNAs, two types of self-splicing can be distinguished, the so-called group I and group II introns. Even if it could be conclusively shown that the RNA has the catalytic activity, proteins seem to be additionally involved in the reactions of both groups of introns in vivo , which probably promote the development of the active structure of the RNA. Since the total number of phosphodiester bonds always remains the same in the reactions described below because they are transesterifications , no energy-supplying cofactors are necessary.
Group I introns occur in the pre-rRNA of simple eukaryotes such as eukaryotes. B. the already mentioned ciliate T. thermophila , as well as in some pre-mRNAs of cell organelles such as mitochondria and chloroplasts . The excision of the intron takes place in a two-step mechanism, with a guanosine , which isessential as a cofactor for the reactionand which is brought into the appropriate position by the structure of the RNA, initiallyperforminga nucleophilic attack on the 5'- splice site . The nucleofugal group of this reaction, the 3'-hydroxyl group of the 5 'exon, in turn attacks the 3'-splice site as a nucleophile, as a result of which the two exons are linked to one another, releasing the intron. In subsequent reactions the intron finally closes into a ring. Stereochemical studies on chiral substrates suggest that a single catalytic center catalyzesboth partial reactions of splicing in the form of a back and forth reaction.
Group II introns , on the other hand, are found in pre-mRNAs of the mitochondria of yeast and other fungi and in some RNA precursors of the chloroplasts of some unicellular eukaryotes such as Chlamydomonas . A guanosine cofactor is not necessary here; rather, due to the structure of the RNA, an adenosine 7 or 8 nucleotides upstream of the 3 'splice site isable to attack the 5' splice site nucleophilically with its 2 'hydroxy group can. This leads to the formation of an unusual 2 '5' phosphodiester bond and thus to the formation of a lasso-like structure of the intron, the so-called lariat . In a second reaction, similar to that of the group I introns, the 5 'splice site finally attacks the 3' splice site nucleophilically, which leads to the linking of both exons and the release of the intron.
In the spliceosomal processing of mRNAs (see below) a reaction mechanism is found which is identical to that of the group II introns, which has led to a number of speculations as to whether both processes evolved from each other (see below under the “intron early” hypothesis ), for example by fragmentation of a group II intron, or whether it is convergent developments due to the catalytic optimization of the same chemical reaction.
Protein splicing is defined analogously to splicing of RNA .
Splicing of tRNAs
The enzymatic splicing of tRNAs can be found in both archaea and eukaryotes , whereas in bacteria introns in tRNAs are processed according to an autocatalytic mechanism that was described in the previous section. The introns in the genes coding for the tRNAs are usually found in the anticodon loop directly 3 'of the anticodon - more rarely in the dihydrouracil loop - and are 14 to 60 bases in length. In the case of enzymatic splicing, in contrast to the splicing of pre-mRNAs, they are not recognized by their sequence but by a higher-level structure of the overall molecule (for example the bulge-helix-bulge-motif - BHB-motif - in archaea) and removed in three steps. The pre-tRNA is first cut twice by an endonuclease , which releases the intron and two so-called tRNA half molecules. The resulting cyclic 2 '3' phosphate of the 5 'half-molecule is then hydrolyzed to a 2' phosphate and a 3 'OH group, while the 5' OH group of the 3 'half-molecule is phosphorylated with GTP consumption. This enables ligation by an RNA ligase with ATP hydrolysis. Finally, in the last step, the 2 'phosphate is removed, which, unusually, takes place with NAD consumption and the release of nicotinamide. Some mRNAs are also processed according to a similar mechanism, which is actually very atypical for them, consisting of two endonucleolytic cleavages with subsequent ligation by a tRNA ligase.
Splicing in the spliceosome
In most cases, splicing takes place in a large complex of RNA and proteins, the so-called spliceosome , which catalyzes the reaction in two successive transesterifications . The majority of the introns are removed in this way. The total number of bonds remains the same during the reaction, energy is only required for the construction and rearrangement of the machinery for catalysis (spliceosome). The two individual reactions do not differ chemically from one another, only the positions of the groups involved in the pre-mRNA are different. In both reactions, a nucleophilic substitution (S N 2) takes place on a phosphate , the nucleophile is in each case a hydroxyl group of a ribose .
In the first step, the oxygen atom of the 2'-OH group of an adenosine from the so-called "branch point sequence" (BPS) attacks a phosphorus atom of a phosphodiester bond in the 5'-splice site. This leads to the release of the 5 ' exon and to the circularization of the intron (called “lariat” due to the lasso-like structure). In the second step, the oxygen of the released 3'-OH group of the 5'-exon attacks the 3'-splice site, which leads to the linking of the two exons and the release of the intron lariat.
The splicing pattern can differ due to the type of fabric and environmental influences. One speaks of alternative splicing , an important basis for a great diversity of proteins. Splicing takes place cotranscriptionally, which means that introns are already removed while the polymerase is reading the gene.
Other important processes that occur during the maturation of a pre-mRNA to mRNA are
- Capping : Modification of the 5 'end of the RNA with a 7-methylguanosine for better stability of the RNA and important for translation on the ribosome .
- Tailing : After reaching the end of the gene, the RNA is cut around 15 nucleotides according to a special base sequence ( AAUAAA ) and given a poly-A tail that is around 150-200 nucleotides long . Here, too, a large number of proteins play a role (CPSF complex, Cstf complex, CFI, CFII, PABP2, PAP etc.), which, in addition to the A2UA3 sequence mentioned, bind other elements of the RNA and regulate processing. A termination of the transcription - unfortunately a very little understood process in eukaryotes - takes place a little later downstream of the polyadenylation site and the like. a. through the TREX complex.
Finally, the mature mRNA is through the nuclear pores (nuclear pore complex, NPC) from the nucleus to the cytosol exported where it then, in the course of the translation is used to proteins to synthesize.
Splicing and Diseases
Splicing also plays a major role in some clinical pictures. Mutations in introns have no direct effect on the sequence of the protein encoded by a gene. In some cases, however, mutations affect sequences that are important for splicing and thus lead to incorrect processing of the pre-mRNA. The resulting RNAs code for non-functional or even harmful proteins and thus lead to hereditary diseases .
A classic example are some forms of β- thalassemia , a hereditary hemoglobinopathy in which a point mutation changes the 5 'splice site of intron 1 of the HBB gene and thus makes it unusable. This leads to nearby “cryptic” splice sites being recognized and the spliceosome producing shortened or lengthened mRNAs that are translated into inactive proteins . Another well-studied mutation in intron 2 of the same gene leads to the retention of a short intron sequence in the final mRNA. In both cases there is a greatly reduced hemoglobin synthesis of the allele in the erythrocytic precursors. If both alleles are affected by such a mutation, the clinical picture of β-thalassemia major arises , which u. a. leads to significant anemia and a constant need for transfusions .
Other cases are e.g. B. Ehlers-Danlos syndrome (EDS) type II (mutation of a branch point in the COL5A1 gene ) and spinal muscular atrophy (mutation of a splicing enhancer / silencer in the SMN 1 gene ).
The "RNA Factory"
In recent years it has become increasingly clear that transcription , processing of RNA (i.e. splicing, capping and tailing ), RNA export into the cytoplasm , RNA localization, translation and RNA degradation influence and regulate each other. The processing of the pre-mRNA takes place during the transcription - one speaks of cotranscriptional RNA processing - and the different machines make contact with each other. For this reason, the term “RNA factory” was recently coined to illustrate this. The splicing can also influence the processes that take place in the cytoplasm in separate locations. A protein complex that is deposited on the finished mRNA by the spliceosome (the exon junction complex, EJC) enables effective export from the cell nucleus and additionally transmits information that enables later quality control of the RNA during translation ( nonsense-mediated mRNA decay , [NMD]). Another implication that arises from this is: a complete pre-mRNA (as shown in the figure above) does not actually occur in the living cell, because introns are removed during transcription, as just described.
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 that encode such protein domains brings with it the possibility of using a domain that has once been “invented” in an evolutionary manner by combining it with others. By simply recombining exons according to a modular principle, a large variety of proteins with a wide variety of functions and properties can be created, which is known as exon shuffling . A classic example of this is the gene for the protein fibronectin, which on the one hand plays a role in cell adhesion and, on the other hand, also in cell migration, proliferation and differentiation. The protein mainly consists of repetitions of three protein domains, which can also be found in the plasminogen activator protein (type I), in blood coagulation proteins (type II), cell surface receptors and proteins of the extracellular matrix (type III).
In addition, there are assumptions that introns could already have been present in the last common, universal ancestor ( last universal common ancestor , an organism from which the three kingdoms of bacteria, archaea and eukaryotes developed). This intron early hypothesis is supported by the discovery of different introns in the genomes of mitochondria, archaea and viruses. According to this theory, bacteria must have lost their introns, which could be explained by optimizing the genome for rapid proliferation and short generation times. In contrast, at least some of the introns do not seem to conform to this theory, as they are believed to have evolved from other precursor sequences. Accordingly, there may not have been a “primordial intron” (from which all today's introns arose), but rather several sequences as ancestors for the introns known today. Thus introns would not be monophyletic , but would most likely correspond to a polyphyletic group. This relationship is formulated in the intron-late hypothesis.
See also
literature
- James E. Darnell , Harvey Lodish, David Baltimore : Molecular Cell Biology . de Gruyter, Berlin et al. 1993, ISBN 3-11-011934-X (4th edition. Harvey Lodish: Molecular Cell Biology. Spectrum Akademischer Verlag, Heidelberg et al. 2001, ISBN 3-8274-1077-0 ).
- Benjamin Lewin: Molecular Biology of Genes . Spektrum Akademischer Verlag, Heidelberg et al. 1998, ISBN 3-8274-0234-4 .
- William S. Klug, Michael R. Cummings, Charlotte A. Spencer: Genetics. 8th, updated edition. 2007, ISBN 978-3-8273-7247-5 .
Web links
- mRNA splicing (animation) from Alberts et al., Essential Cell Biology.
Individual evidence
- ^ "Nobel Prize in Medicine, 1993" Official website of the Nobel Prize Committee. Retrieved June 18, 2010.
- ^ "Nobel Prize in Chemistry, 1989" Official website of the Nobel Prize Committee. Retrieved June 18, 2010.
- ↑ Sebastian M. Fica, Nicole Tuttle, Thaddeus Novak, Nan-Sheng Li, Jun Lu: RNA catalyses nuclear pre-mRNA splicing . In: Nature . tape 503 , no. 7475 , November 14, 2013, ISSN 0028-0836 , p. 229–234 , doi : 10.1038 / nature12734 , PMID 24196718 , PMC 4666680 (free full text) - ( nature.com [accessed May 5, 2016]).
- ↑ Raffaella Origa: Beta Thalassemia . In: GeneReviews® . University of Washington, Seattle, Seattle (WA) 1993, PMID 20301599 ( nih.gov [accessed April 5, 2020]).
- ↑ Antonio Cao, Renzo Galanello: beta-thalassemia . In: Genetics in Medicine . tape 12 , no. 2 , February 2010, ISSN 1530-0366 , p. 61–76 , doi : 10.1097 / GIM.0b013e3181cd68ed ( nature.com [accessed April 5, 2020]).