The stages of protein synthesis include transcription, mRNA processing, mRNA splicing and translation. Pre-mRNA splicing is the removal of introns, which are non-coding sequences for genes and essentially non-sense codes found on pre-mRNA strands, staggered within coding sequences of genes called exons. All mRNA strands involved in protein synthesis must undergo pre-mRNA splicing in order to produce mRNA that is then ready to undergo translation so that functional proteins can be produced.
There is a substantial difference in the length of introns and exons with introns being much longer ranging from approximately 80 nucleotides to 10,000 nucleotides and therefore a considerable amount of RNA can be excised in the process of splicing.
The gene coding for the specific protein being produced by the cell is transcribed into a pre-mRNA strand known as the primary transcript. A methylated cap is added to the 5' end of the strand. The 3' end of the strand is then cleaved and a polyadenylated tail is added. This part of the process is known as mRNA processing precedes splicing.
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Splicing is carried out by a complex of proteins and RNA assembled together known as small nuclear ribonucleoprotein particles (snRNPs) collectively giving rise to the spliceosome. The important locations for splicing to be carried out on the primary transcript include a GU 5' donor site, 20-50 base pairs upstream from this, a branch point A site and an AG 3' acceptor site. These locations are present between exon 1 and exon 2.
The initial step of splicing involves an snRNP called U1 which recognises and binds to the 5' end of the RNA whilst U2 will recognise and bind to Branch point A. More snRNPs will then bind to the U1 and the U2 and form the spliceosome which causes the RNA strand to loop around. As the mRNA strand loops around on itself, the 5' end of the RNA is cleaved and binds to the A branch point forming a lariat in the process. Thus the newly formed 3' site of exon 1 is then able to react with the acceptor splice site by cleaving it and ligating with the second exon.
The excised intron will then go on to be degraded whilst the newly joined exons can be translated into functional proteins.
Two other types of proteins may be involved in the process of splicing known as repressor proteins or activator proteins. These proteins bind either to the splicing silencer site in the case of the repressor protein which represses the notion of the site being used in splicing or in the case of the activator protein, which will bind to the enhancing site so that the site will more likely be spliced. Depending on their location and function, they will be known as either intronic or exonic splicing enhancers or silencers and may also be referred to as Cis-elements.
Alternative splicing is a mechanism by which splicing occurs but is not conventional in that an extra intron may be maintained after splicing, an exon may be missing after splicing or splice sites may vary themselves, eventuating a missing exon or extra intron. However, alternative splicing may still lead to viable proteins. In some cases however spliced genes can account for promoting or inhibiting apoptosis e.g. the Bcl-X gene which regulates cell apoptosis, if incorrectly spliced, can lead to invasive human breast carcinomas as well as being associated with various lymphomas (Eurasnet).
There are in fact many human diseases associated due to incorrect splicing including many cancers, a few of which include tumours of the breast, colon, nervous tissue, kidney and many more (Sanger institute).
BRCA1 is a breast cancer gene which has been reported to be connected with splicing mutations. A study performed in 2003 (Yang et al) found a new 5' splice site formed during protein synthesis from a substitution mutation of a nucleobase C to G in the BRCA1 gene, which resulted in a non-functioning protein, whilst also interfering with the function of the splicing enhancer. This particular study also investigated mutations of other codons and attributed these mutations to the role of the affected cis-elements.
A separate study in 2004 (Sharp et al), looked at even more genes that possible underwent splicing mutations including BRCA1, BRCA2 and MLH1 associated with colon cancer. In the case of the BRCA1 gene, the study found that there was loss of expression of this gene from unknown causes but needed more studies to cement conclusions however, the same study also found mutations in splicing including exons being missed out and parts of introns, being retained during splicing.
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Cystic fibrosis is a respiratory disease which arises from mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene on Chromosome 7. This results in a chloride channel defect of secretion and an uptake in Sodium absorption in the respiratory tract. A patient with cystic fibrosis suffers from repeat chest infections and respiratory problems, GI symptoms such as pancreatic insufficiency, cirrhosis and gallstones as well as others such as nasal polyps and male infertility.
Hefferon et al conducted research in which the upstream 5' splice site underwent a change in purine which gave rise to an increased probability of exon 9 in particular to be contained in the newly synthesised mRNA strand, post splicing. Interestingly, altering the downstream 5' splice site, with respect to this study, inserting adenine at this location, the probability of this exon being missed out in splicing greatly decreased.
SMA (spinal muscular atrophy) is a disease which arises from a defect or exclusion of the SMN1 and SMN2 genes. It is a neurodegenerative disorder in which patients suffer from wasting of the muscles of the limbs and weakness.
It is commonly caused by a defective SMN1 gene such that an snRNP's activity is diminished on a large scale. SMN2 genes however, may constitute a C to T base substitution in exon 7, possibly due to the loss of an exon splicing enhancer or the production of an exon splicing silencer. This exon will then be skipped in splicing. The result is a very low level of a functional protein.
A study in 2007 (Marquis et al) looked at correcting this splicing mutation using an snRNA (small nuclear ribonucleic acid) to provide a complimentary antisense sequence which could bind to exon 7 as well as an exon splicing enhancer. The study concluded that it was possible to correctly splice all SMN2 genes.
Thalasaemia is a disorder in which there is an uneven balance of haemoglobin synthesis, varying from either no production of a chain to a reduced production of the globin chain. This causes an array of symptoms from anaemia, splenomegaly, failure to thrive, osteopenia in association with β thalasaemia to moderate anaemia, leg ulcers, jaundice and possibly even death in utero with respect to α thalasaemia.
Once again, owing to a point mutation which then lead to exon skipping and part of an intron being retained and the formation of a new splice site, had resulted in α thalasaemia-myleodysplastic syndrome in one individual (Nelson et al).
Even much older research (Merill et al) found that a substitution mutation gave rise to an abnormal 5' splice site however retained the normal 3' splice site but resulted in a very defective β globin.
Neurofibromatosis type I is a neurological disorder in which a patient exhibits dermal neurofibromas, nodular neurofibromas, Lisch nodules on the iris, Cafe-au-lait spots on the skin and freckling. Complications may arise from GI bleeds, cystic lesions of the bone, scoliosis or even pheochromocytoma.
Wimmer et al describes a study in which a large percent of the patients had an underlying mutation where 38% of these mutations were due to splicing mutations and sequence alterations causing exon skipping, exons which should not have been included, formation of new splice sites leading to loss of exons and splice site disruption creating new splice sites.
There are many more diseases for which mutations in splicing are to blame but as these are now well known, it is the correction of these mutations that are of interest in order to combat the diseases such as cancer (Ghigna et al).
Similarly to the procedure mentioned for the correction of the splicing mutation for SMA, antisense morpholino oligonucleotides are being used to correct splicing mutations in that of the ataxia telangiectasia mutated gene by repressing cryptic splice sites (Liutao Du et al). The same compounds have been used successfully in the treatment of the CFTR gene, β globin gene and Dystrophin gene and the ongoing research and development of identifying and correcting of splicing continues (Tosi et al).