Splicing Mech

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 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.

GUUGGUAU

CACUGAC

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

+15

-30

5.8S

Transcribed Intragenic spacer

Start point

Pre RNA gene

28S

Intergenic spacer

tRNA genes

Eukaryotic tRNA gene

30 – 60 bp

Transcribed spacer

Promoter block A 11 bp

Intergenic spacer

+8 Start point

+19

Promoter block B 9bp

Transcribed spacer

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.

G

Exon1

Exon2

P

P

G

PPP

OH

Intron

Exon2

P

OH

PPP

P

Exon1

P Exon1

Self catalysed Intron Splicing in rRNA

Exon2

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

Exon1

Exon2

P

P

Intron RNA endonuclease

OH

OH

P

P

Exon2

Kinase RNA ligase Exon1 Cyclic phosphodiesterase

P

P

P OH

P Exon1

P

Phosphatase

OH

Exon1

Exon2

P Exon1

Exon2

Exon2