RNA SPLICING.

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In Eukaryotes, the RNA transcribed from DNA almost invariably undergoes RNA splicing to yield mature RNA sequences. It involves removal of sequences mainly corresponding to introns in split genes. The mechanisms available for this purpose include the following:

        I.      Self splicing of fungal mitochondrial and other group I introns;

     II.      Splicing of higher eukaryotic nuclear introns, through the formation of spliceosomes at intron-exon junction;

  III.      Self splicing of mitochondrial group II introns through lariat formation without assistance from any proteins or spliceosomes;

  IV.      Splicing of yeast tRNA precursor molecules by cleavage due to endonuclease followed by fusion due endonuclease followed by fusion due to ligase.

Self-splicing of RNA molecules involving group I introns, found in rRNA genes of Tetrahymena and Physarum nuclei, in fungal mitochondria and in phage T4, takes place through two transesterification relations. Group I introns are characterized by (i) the absence of conserved sequences at splicing junction, and by (ii) the presence of short conserved consensus sequences internally. In the first transesterification, the 5’ splice site is cleaved. In the second transesterification step, the 3’ splice site is cleaved. The excised IVS or introns can form a circle by cyclization reaction and these circles can again regenerate linear molecules due to autocatalysis.

In case of viroids and virusoids= satellite RNA, a consensus sequence forms a ‘hammerhead’

SPLICING OF hnRNA OF HIGHER EUKARYOTES THROUGH SPLICEOSOMES.

Splicing of major class of GU-AG introns. Splicing of introns sequences of eukaryotic hnRNA involves a well defined multi-step pathway. Small nuclear ribonucleoprotein particles and about 50 protein factors are essential for the formation of an active spliceosomes, in which introns excision proceeds in two successive transesterification reactons. Each step of the splicing reaction is mediated by a number of snRNA associated proteins and non-snRNP splicing factors. The major components of the splicing machinery in mammals, which may differ in details worked out in budding yeast. (i)snRNPs consisting of snRNAs and common Sm proteins, (ii) SR family of splicing proteins; (iii) polypyrimidine tract-binding proteins; (iv)branh-site binding proteins; (v)hnRNP proteins; (vi) snRNP associated non-snRNP proteins; (vii) Some other non-snRNP splicing factors.

THE SPLISOSOME ASSEMBLY: The splicing reaction involves the formation of spliceosome. For spliceosome assembly. The splicing reation involves the formation of spliceosome. For spliceosome associate with pre-nRNA using large number of essential protein factor.

1)      SR proteins: They are called SR proteins serine, abbreviated as S and arginine abbreviated as R; the N-terminal region consists of an RNA recognition motif or RRm. SR proteins are among the first components that interact with pre-nRNA, thus committing pre-nRNA to the splicing pathway.

2)      Polypyrimidine tractbinding proteins; They bind a polypyrimidine tract and play an important role in spliceosome assembly. Following steps are involved in spliceosome assembly (i) The first specific stage of spliceosome assembly is the binding of U1 sn RNP, which requires interaction of pre-mRNA with SR.

Splicing of UA rich introns in plants. Plant introns have UA-rich elements spread throughout their length. These UA-rich elements help in recognition of 5’ and 3’ splice sites.

Following interactions are involved in plant intron recognition:

(i)                 U1 snRNP binds to 5’ splice site,

(ii)              U-tract binding factors bind the U-rich sequence preceeding the 3’ splice site,

(iii)            UA-island binding proteins bind to UA-rich elements of intron and

(iv)             exon sequence element binding proteins associate with AG elements in the adjacent exon. In plants, splice site selection is primarily defined by UA- rich sequences within the intron. Consequently UA richness in plant introns is essential for efficient splicing and for 5’ and 3’ splice site recognition. However, the monocot splicing machinery is more permissive than the dicot recognition machinery.

Formation of lariat during splicing. Nuclear splicing, where spliceosome is formed, involves formation of a lariat structure. This occurs in two stages: (i) In the first stage, a cut is made at the left end of the intron, releasing a separate RNA molecule with left exon and a right RNA molecule with intron and the right exons.

The left RNA molecule is linear, but the right intron-exon molecule is not linerar The 5’ terminus at the left end of intron-exon molecule gets liked by a 5’-2’ bond to the A of the sequence CUGAC located-30 bases upstream of the right end of the intron. This linkage generates a lariat. (ii) In the second stage, cutting at the right splicing junction releases a free intron in lariat form, and the left exon is ligated to the right exon. The lariat is debranched to give a linear excised intron, which is rapidly degraded.

Splicing of group II introns

The group II introns resemble introns of nuclear pre-mRNA or hnRNA of higher eukaryotes and are excised as lariats like those produced in nuclear pre-mRNA introns of higher eukaryotes. They have consensus sequences at the splicing junctions, GT and AP_y  and a branch sequence resembling TACTAAC box. In group II introns, higherly conserved sequence elements juxtapose splice sites and branch sites by intramolecular base pairing interactions, in the nuclear pre-mRNA introns, the same functions are compensated by intermolecular interactions between the RNA substrate and the RNA moieties of snRNPs. This self splicing reaction of group II introns may be regarded as an intermediate step between RNA mediated self splicing in group I introns and protein dependent RNA splicing of the introns of nuclear pre-mRNA of higher eukaryotes.

Yeast tRNA splicing by cutting and  rejoining

About 40 genes of appro. 400 genes for yeast nuclear rRNAs are interrupted, each with a single intron, located one nucleotide away from 3’ end of anticodon. The splicing requires following two steps (i)phosphodiester bond cleavage by an endonuclease; this does not require ATP; and (ii) ligation reaction, which requires ATP and involves bond formation with the help of RNA ligase;

Constitutive vs alternative splicing

In the earlier sections of this chapter, we have discussed the various mechanisms involved in RNA splicing. Generally, the pre-mRNAs undergo processing, so that the non-coding intervening sequences, i.e. introns, are excised, and exons correctly ligated. When this splicing takes place in all cells, without any variation, it is described as ‘constitutive splicing’ in contrast to ‘alternative splicing’, which differs in time and space. Thus ‘alternative splicing’ is an adjunct to the regulation by promoter activity, gene rearrangements and by the occurrence of multigene families. In alternative splicing, splice sites that are selected for splicing in some circumstances are completely by-passed by the splicing machinery under other circumstances. The latter may lead to (i) insertion of a peptide segment or, (ii) functionally different product or (iii) no functional product.

Ribozymes (RNA splicing, DNA Cleavage and RNA Amplification)

RNA molecules can cut, splice and assemble themselves without any outside help, thus extending the range of chemistry of enzymes. These RNA molecules working as enzymes, were called ribozymes. It has also been shown that enzymes can also be synthesized chemically, and are then described as chemzymes.