4 major types of introns

• 4 classes of introns can be distinguished on

the basis of their mechanism of splicing
and/or characterisitic sequences:
– Introns in pre-tRNA
– Group I introns in fungal mitochondria, plastids,
and in pre-rRNA in Tetrahymena (self-splicing)
– Group II introns in fungal mitochondria and
plastids (self-splicing)
– Introns in pre-mRNA (spliceosome mediated)
Table 31-4 Types of Introns.
Co-Transcriptional RNA Processing
• Remember, the phosphorylated C-terminal domain
(CTD) of RNA polymerase II binds to enzyme complexes
involved in RNA processing.
– Both addition of the 5’ cap and the splicing of introns.
– The basic CTD sequence is repeat with the consensus Y-S-P-T-S-P-S
– The CTD is absent or very different in some putatively primitive
eukaryotes (e.g., Giardia, Trichomonas, trypanosomes).
The Splicing Reaction
• An unusual 5’-2’
linkage is made
between the
CACUGAC
GUUGGUAU

branch point
nucleotide and the
5’ splice site.
• The the free 3’ end
of the 5’ exon will
link to the 3’ splice
site.
• This liberates the
so-called lariat
structure, which is
degraded.
The Splicing Reaction
• An unusual 5’-2’
linkage is made
between the
branch point
nucleotide and the
5’ splice site.
• Notice that this
generates a lasso
or lariat structure,
which is initially
attached to the 3’
splice site.
• The free 3’ end of
the 5’ exon will
undergo a second
transesterification.
The Splicing Reaction
• The free 3’ end of
the 5’ exon will
undergo a second
transesterification.
• This liberates the
lariat structure.
Figure 31-49 The sequence of
transesterification reactions that splice
together the exons of eukaryotic pre-mRNAs.
 Splicing of pre-mRNA
• The introns begin and end with almost invariant
sequences: 5’ GU…AG 3’
• Use ATP hydrolysis to assemble a large spliceosome
(45S particle, 5 RNAs and 65 proteins, same size
and complexity as ribosome)
• Mechanism is similar to that of the Group II fungal
introns:
– Initiate splicing with an internal A
– Uses a phosphoester transfer mechanism for
splicing
Initiation of phosphoester transfers in
pre-mRNA
• Uses 2’ OH of an A internal to the intron
• Forms a branch point by attacking the 5’
phosphate on the first nucleotide of the intron
• Forms a lariat structure in the intron
• Exons are joined and intron is excised as a
lariat
• A debranching enzyme cleaves the lariat at
the branch to generate a linear intron
• Linear intron is degraded
 rRNA Genes

Transcribed Intragenic spacer

Promoter 18S 5.8S 28S

+15
-30 Transcribed Intragenic spacer Intergenic spacer

Start point

Pre RNA gene

 tRNA genes

Eukaryotic tRNA gene

30 – 60 bp

Promoter block A Promoter block B

Transcribed spacer 11 bp 9bp Transcribed spacer

Intergenic spacer

+8 +19

Start point
 rRNA splicing - self catalysis

Guanosine acts as a cofactor.

Guanosine binds to the pre-rRNA at the 5’ end of the intron. binding at this
point is promoted by the secondary structure of the intron.

Attachment of the GTP and the conformational change favor the transfer
of the phosphate group at the 5’ boundary of the intron to GTP.

The role of GTP is to provide a 3’ OH group for attachment of the 5’ end of

the intron.

The phosphate group at the 3’ boundary of the intron is transferred to the

–OH group at the 3’end of the exon 1.
 P Exon2
P
Exon1
G

Intron

OH
PPP

P Exon2
P
PPP

OH

Exon1

P Exon2

Exon1

Self catalysed Intron Splicing in rRNA

Splicing mechanism of tRNA
Breaks are made by an RNA endonuclease at the 5’ and 3’ ends of the
intron

The cut at 5’ end leaves a cyclic 2’3’-phosphate group exposed at the end
of the exon

The cleavage at the 3’end leaves a 5’OH exposed on the exon

The cyclic bond is opened by cyclic phosphodiesteraseat the 3’end of the

exon at the 5’ splice site

A phosphate is added at the 5’end of the exon at the 3’ splice site by a

kinase.

RNA ligase then reacts with an ATP to ligate the two exons.

A phosphatase then removes the 2’phosphate from the ligation site.

 Intron Splicing in tRNA

P Exon2
P
Exon1 Intron

RNA endonuclease

OH
Exon2

OH
P
Kinase
RNA ligase
Exon1
Cyclic phosphodiesterase

P Exon2
P
P
P Exon2
OH

Exon1 Exon1 P
Phosphatase
OH

P Exon2

Exon1