In bacteria, the process of transcription and translation takes place simultaneously so the polypeptide sequence that is encoded by DNA in bacteria is collinear with its amino acid sequence. But this is not true in the case of eukaryotic mRNAs. In 1977 Phillips Sharp and Richard Roberts noticed that many eukaryotic genes are interrupted by noncoding sequences known as introns and coding sequences present between them are termed as exons. Most of the eukaryotic genes and archaebacterial genes contain introns except genes that encode for histones and genes present in Saccharomyces cerevisiae.
During transcriptional process, both exons and introns are transcribed from gene by RNA polymerase but introns are removed from primary RNA transcripts. This removal process is termed as RNA splicing. RNA splicing process in which introns are spliced and exons are joined to produce mature mRNA, is completed through two transesterification reactions.
Types of Introns
There are four classes of introns which are described below:
Group I introns: Group I introns are found in some nuclear, mitochondrial and chloroplast genes coding for rRNA, mRNA, and tRNA. They are self-splicing i.e. they does not require enzyme for intron splicing.
Group II introns: Group II introns are present in mitochondrial and chloroplast mRNA of fungi, algae, and plants. Like Group I introns, they are also self-splicing.Both Group I and Group II introns do not require ATP in their splicing mechanism.
Group III introns: Group III introns are found in nuclear mRNA primary transcript of eukaryotes. They require energy in form of ATP and the removal of introns is facilitated by a large protein complex known as spliceosome. So, these introns are also known as Splicesomal introns.
Group IV introns: Group IV introns are generally seen in certain tRNAs. The splicing action to remove these introns needs ATP and endonuclease.
In this article we will mainly focus on splicing mechanism of Group III introns or spliceosomal introns.
Structure of Spliceosome
As mentioned above that this group of introns is spliced by a protein complex Spliceosome. Spliceosome is made up of specialized RNA- protein complexes known as small ribonuclear proteins (snRNPs). Each snRNPs consists of one of the class of RNAs which is supposed to be 100-200 nucleotides long, known as small nuclear RNAs (snRNAs). There are five snRNAs U1, U2, U4, U5 and U6 known to be involved in splicing reaction.
Splicing Mechanism of Group III introns
Spliceosomal introns have the GU nucleotide at 5'end known as 5'splice site and AG nucleotide at 3' end known as 3'splice site. Along with 5' splice site and 3' splice site introns are also seen to contain a branch point site containing A nucleotide. U1 snRNP contains sequence complementary to sequence of the 5'splice site of mRNA of intron and U1 snRNPs bind to complementary region of primary transcript which helps in defining 5'splice site during spliceosomal assembly. U2 snRNPs is paired to introns at branch point site encompassing A nucleotide that act as nucleophile during splicing reaction. At each step energy in form of ATP is required by spliceosomal complex. Pairing of U2 snRNPs to introns leads to the formation of a bulge that displaces A nucleotide whose 2'OH group acts as a nucleophile.
Binding of U1 and U2 snRNPs are followed by binding of U4/U6 snRNPs and U5 snRNPs. The next event in splicing process is expulsion of U1 and U4 snRNPs from spliceosomal complex and U6 gets paired with 5'splice site of intron forming lariat shaped structure as an intermediate. This contains an unusual 2'-3'phosphodiester bond along with the usual 3'-5' phoshodiester bond. 3'OH of 5' exon nucleophillically attack on the 3'end of intron which results into precise excision of intron and ligation of exon to form mature mRNA.
Significance of Splicing
Most of the eukaryotic mRNAs are translated to produce one corresponding polypeptide chain but some transcripts have alternative splicing pattern i.e. they can be processed in more than one way to produce different mRNAs and sequentially different type of protein products. This form of splicing is termed as alternative splicing. A beautiful example of alternative splicing is possessed by fruit fly, at its different stage of development three different forms of myosin heavy chains are formed by common primary transcript.
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