Let us make an in-depth study of the split genes or interrupted genes. After reading this article you will learn about: 1. Processing of mRNA by Splicing 2. Spliceosome 3. Mechanism of Splicing 4. Self Splicing and 5. Alternate Processing of Pre-mRNA.
Processing of mRNA by Splicing:
Till recently it was believed that coding sequence of DNA and amino acids of polypeptide is collinear. The coding sequences are continuous and codon for one amino acid is adjacent to the codon for the next amino acid. The open reading frame is a single stretch of codons without any gap.
But now it has been discovered that coding sequence of most of enkaryotic genes is split into stretches of codons interrupted by stretches of non-coding sequences. Most human genes are discontinuous.
The coding sequences of DNA of the gene are called exons. In between exons, there are intervening non-coding sequences called introns. This type of genes are called split genes or interrupted genes. They are most common in enkaryotes. They are also found in viruses but rarely in bacteria.
The terms exons and intons were given by Gilbert in 1977. It was discovered in Amphibia, mammals and some other animals that genes are not represented by continuous sequence of nucleotides. Introns are removed by excision and discarded.
The size of introns and exons varies Introns are usually much larger than the exons. A typical exon consists of small number of nucleotides whereas an intron may consist of thousands of nucleotides. Moreover, the introns constitute a large portion of the genome.
During the processing of mRNA in enkaryotes, the amount of discarded RNA ranges from 50- 90 percent of the primary transcript. The remaining segments of mRNA or exons are joined together to form the finished mature mRNA. The split genes are transcribed into a single mRNA copy of the entire gene, called primary transcript. It consists of both exons and introns.
The removal of introns by excision is called RNA splicing. The 5′-segment or the cap and the 3′-segment of the trailer are never discarded. Some examples of number of introns are — a- globin has two introns. Yeast genes have one intron, ovalbumin has seven, a-collagen has 52 introns and Titin gene of human beings has 363 introns.
Translation takes place only after the splicing is completed.
Phillip Sharp and Richard Robert won Nobel Prize for medicine in 1993 for their work on split genes. They studied the hybrid of adenovirus mRNA and one (template) strand of DNA under the electrons microscope. The mRNA was found to hybridize with discontinuous stretches of genomic DNA. The intervening stretches of DNA were in the form of loops. These loops represented introns.
The splicing by excision is remarkably precise and accurate and cuts or nicks are made at highly precise position. Because even if an error of one base occurs, the correct reading frame would to disturbed and wrong amino acid will be coded. Chambon studied the boundaries of the introns.
The end of intron always have GU towards the 5′-end (5′-GU-3′) and AG towards the 3′-end (5′-AG-3′). This is known as GU-AG rule or chambon’s rule according to which an intron begins with GU and ends with AG. The boundary between exon-intran at 5′-end is called 5′-splice site, while boundary between intron-exon at 3′-end is called 3′-splice site.
Introns are excised one by one. The AG at the 3′-end is preceded by a pyrimidine rich sequence called polypyrimidine tract (Py tract). About 10-40 bases upstream of the polypyrimidine tract is a sequence called branchpoint sequence (A), which is 5′-UACUAAC- 3′ in yeast. This sequence is required in lariat formation during splicing process.
RNA Splicing:
Splicing is generally performed by endonuclease enzymes cleaving the introns at both ends. Phosphodiester bond between sugar and phosphate at the junction between intron and exon is cleaved. The freed 5′-end of the intron joins the branch point sequence of form lariat.
Spliceosome:
Splicing is performed by a large complex called spliceosome. The spliceosome is made up of small nuclear ribonuclear proteins (sn RNP) called snurps. These consist of RNAs which are rich in uracil and are of several types U1, U2, U4, U5 and U6 which are collectively called small nuclear RNAs (sn RNA).
Mechanism of Splicing:
At first U1 sn RNP recognize and-break the 5’splice site of the intron and bring it closer to branch site. Then the complex of sn RNP of U2, U4, U5 and U6 bind to the intron. The complex of snRNPs and precursor mRNA of the intron is called spliceosome. The spliceosome is looped out. This loop of intron is called lariat which is discarded and degraded. The exons on either side of the removed intron are brought closer and ligation seals them together.
Self Splicing:
In some cases specially group I introns, the intron itself folds and catalyzes its own splicing. Here RNA of the intron functions as an enzyme and behaves like an endonuclease to splice out the intron. The RNA which acts as an enzyme is called ribozyme. There the splicing of the intron sequences requires no other enzyme. The intron is released in a linear form which is subsequently degraded.
Alternate Processing of Pre-mRNA:
Primary transcript (pre-mRNA) generally exhibits one pattern of RNA splicing to produce a single type of mature mRNA. But in higher eukaryotes, pre-mRNA exhibits alternate or differential processing patterns to yield different mRNAs containing different exons which encode different proteins. One pre-mRNA after alternate splicing produces more than one type of mature mRNAs.
1. Alternate Splicing:
Here a pre-mRNA has five exons. By alternate splicing it produces two different kinds of mature mRNAs both containing four exons. One type of mRNA consists of exons 1, 2, 4 and 5 while the other consists of exons 1, 2, 3 and 5.
2. Exon Skipping:
Here a pre-mRNA has three exons. Splicing process removes exon no. 2, while exons 1 and 3 are fused and sealed, in the mature mRNA.
3. Intron Retaining:
Here a pre-mRNA has three exons and two introns. Splicing process removes the intron no. B while intron no. A is retained in the mature mRNA. It is possible to retain only a part of an intron.