Chapter 13Chapter 13RNA SplicingRNA Splicing

ByMohammed Z. Naji

Introduction The number of introns found within a gene

varies from one in the yeast genes (and a few human genes), to 50 in the case of the chicken proᾳ2 collagen gene

to as many as 363 in the case of the Titin gene of humans. Also, the sizes of the exons and introns vary.

a typical eukaryotic gene in which the coding region is interrupted by three introns, splitting it into four exons

Introduction introns are very much longer than the exons. for

example, exons are order of 150 nucleotides, whereas can be as long as 800,000 nucleotides (800 kb).

other example, the mammalian gene for the enzyme dihydrofolate reductase is more than 31 kb long, and contain six exons that correspond to 2 kb of mRNA.

Thus, in this case, the coding portion of the gene is less than 10% of its total length.

Introduction In the extreme case of the human

dystrophin gene, RNA polymerase must traverse 2,400 kb of DNA copy (entire gene) into RNA. (transcription rate is 40 nucleotides per second, it require 17 hours to make a single transcript of this gene!)

Alternative splicing, or differential splicing, is mean a single gene coding for multiple proteins.

60% of the genes in the human genome are spliced in alternative ways to generate more than one protein per gene.

For extra example, Drosophila gene & the Slo gene from rat which encodes a potassium channel expressed in neurons has the potential to encode 500 alternative versions of that product.

THE CHEMISTRY OF RNA SPLICING Sequences within the RNA Determine Where Splicing occurs

the exon-intron boundary at 5' end is marked by a sequence called the 5' splice site.

The intron-exon boundary at 3' end of the intron is marked by the 3' splice site. (it was called the donor and acceptor sites)

third sequence necessary for splicing is called the branch point site (or branch point sequence). It is found entirely within the intron, usually close to its 3' end, and is followed by a polypyrimidine tract (Py tract),

The most highly conserved sequences are the GU in the 5' splice site, the AG in the 3' splice site, and the A at the branch site.

the chemistry of splicing achieved by two transesterification reactions in phosphodiester linkages within the pre-mRNA are broken and new ones are form

The first reaction is triggered by the 2 OH of the conserved A at the branch site.

This group acts as a nucleophile to attack the phosphoryl group of the conserved G in the 5' splice site. the phosphodiester bond between the sugar and the phosphate at the junction between the intron and the exon is cleaved and the freed 5' end of the intron is joined to the A within the branch site.

So the 5' exon is a leaving group in the first transesterification reaction

Thus, in addition to the 5' and 3' backbone linkages, a third phosphodiester extends from the 2'OH of that A to create a three-way junction.

In the second reaction, the 5' exon reverses its role and becomes a nucleophile that attacks the phosphoryl group at the 3' splice site This second reaction has two consequences :-

First, and most importantly, it joins the 5' and 3' exons thus, this is the step in which the two coding sequences are actually "spliced" together.

Second, this same reaction liberates the which serves as a leaving group.

Because the 5' end of the intron had been joined to the branch point A in the first transesterification reaction, the newly liberated intron has the shape of a lariat.

In the two reaction steps, there is no net gain in the number of chemical bonds two phosphodiester bonds are broken, and two new ones made. As it is just a question of shuffling bonds, no energy input is demanded by the chemistry of this process.

But, as we shall seebelow, a large amount of ATP is consumed during the splicing reaction. This energy is required, not for the chemistry, but to properly assemble and operate the splicing machinery

is the splicing reaction direction only goes forward?

Two features that could contribute to this are as follows.

First, the forward reaction involves an increase in entropy a single pre-mRNA molecule is split into two molecules, the mRNA and the liberated lariat.

Second, the excised exon is rapidly degraded after its removal and so is not available to partake in the reverse reaction

Exons from Different RNA Molecules Can Be Fused by Transsplicing

In some cases, two exons carried on different RNA molecules can be spliced together in a process called transsplicing. Although generally rare, transsplicing occurs in almost all the mRNAs of trypanosomes. In the nematode worm (C. elegans), all mRNAs undergo transsplicing (to attach a 5' leader sequence), and many of them undergo cissplicing as well.

Trans-Splicing. In transsplicing two exons, initially found in two separate RNA molecules, are spliced together into a single mRNA. The chemistry of this reaction is the same as that of the standard splicing reaction described previously, and the spliced product is indistinguishable. The only difference is that the other product-the lariat in the standard reactionis, in transsplicing, a Y shaped branch structure instead. This is because the initial reaction brings together two RNA molecules rather than forming a loop within a single molecule

THE SPLICEOSOME MACHINERY RNA Splicing Is Carried Out by a Large Complex Called the Spliceosome

The five RNAs (U1, U2, U4, U5, and U6) are collectively called small nuclear RNAs (snRNAs).

Each of these RNAs is between 100 and 300 nucleotides long and is complexed with several proteins.

These RNA protein complexes are called small nuclear ribonuclear proteins pronounced "snurps").

The spliceosome is the large complex made up of these snRNPs, but the exact makeup differs at different stages of the splicing reaction

The snRNPs have three roles in splicing: 1.They recognize the 5' splice site and the branch site 2. they bring those sites together as required. 3. they catalyze (or help to catalyze) the RNA cleavage and

joining reactions. To perform these functions, RNA-RNA RNA-protein, and

protein-protein interactions are all important.

Some RNA-RNA hybrids formed during the splicing reaction. In four cases :

a) different snRNPs recognize the same sequences in the pre- mRNA at different stages of the splicing reaction U1 and U6 recognizing the 5' splice site.

(b) snRNP U2 is shown recog- nizing the branch site.

(c) the RNA:RNA pair- ing between the snRNPs U2 and U6 is shown.

(d), the same sequence within the pre-mRNA is recognized by a protein example,

U2AF (U2 auxillary factor), recognizes the polypyrimidine (Py) tract/3' splice site, and, in the initial step of the splicing reaction, helps another protein,

branch-point binding protein (BBP), bind to the branch site. BBP is then displaced by the U2 snRNP .