Molecular biology of the gene ch 13 rna splicing part 1

1. Chapter 13Chapter 13
RNA SplicingRNA Splicing
By
Mohammed Z. Naji

2. 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.

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

4. 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.

5. 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.

6.  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.

7. 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.

8.  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

10.  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.

11.  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.

12.  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

13.  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

14.  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.

15. 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

16.  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

17.  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.

19. (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
.