BIOL321 - RNA Processing

Cristofre MartinDepartment of BiochemistrySt. George’s University

Comparison of the Structure of mRNA from Prokaryotes and Eukaryotes

1) The 5’ and 3’ end of bacteria mRNA are unmodified.

2) Bacterial mRNA may be polycistronic.

3) Eukaryote mRNA contains a 7-mG cap on the 5’ end and polyadenylation of the 3’ end.

In eukaryotes, the initial product of transcription must be processed and modified to form the mature mRNA.

1) Modification of the 5’ end of the mRNA.

2) Poly-adenylation of the 3’ end of the mRNA (eukaryotes).

3) Splicing of exons and the removal of introns.

RNA polymerase II carries a set of pre-mRNA processing proteins on its tails.

Shortly following the initiation of transcription these proteins are transferred to the nascent RNA.

This transfer of RNA processing proteins typically occurs after approximately 25 nucleotides are produced.

Capping of mRNA: performed by three enzymes.

1) Phosphatase: removes one phosphate from the 5’ end of the RNA.

2) Guanylyl transferase: adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’).

3) Guanine-7-methyl transferase: adds a methyl group to the 7 position of the terminal guanine.

4) 2’-O-methyl transferase: adds a methyl group to the 2’-O position to the next to last base on the 5’ end.

phosphatase

guanylyl transferase

guanine-7-methyl transferase

2’-O-methyltransferase

Capped RNAs are produced on RNA polymerase II transcripts.

The 5’-methyl cap is a characteristic of eukaryote mRNAs and helps the cell distinguish between different RNA in the cell.

The 5’-methyl cap has important roles in the regulation of mRNA processing, transport, and translation.

The modification of the 3’ end of the RNA is accomplished by several enzymes associated with RNA polymerase II that bind to specific sequences on the RNA.

1) Cleavage and polyadenylation specificity factor (CPSF) binds to the hexamer AAUAAA.

2) Cleavage stimulating factor F (CstF) binds the GU-rich element beyond the cleavage site.

3) Cleavage factors bind to the CA sequence at the cleavage site.

CPSF CstF

Poly-adenylation follows cleavage of the 3’ UTR (trailer):

1) Poly-A-polymerase adds approximately 200 A nucleotides to the 3’ end produced by the cleavage.

2) Poly-A Binding Proteins (PABP) binds to the poly-A tail and assist in directing translation by the ribosome.

3) The cleaved fragment of the RNA is degraded in the nucleus.

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Details of intron removal from a pre-mRNA molecule

1) At the 5’ end of an intron is the sequence GU and at the 3’ end is AG (plus some other sequence).

2) Eighteen to 38 nucleotides upstream of the 3’ end of the intron is an A located within the branch-point sequence.

Details of intron removal from a pre-mRNA molecule

3) Intron removal begins with a cleavage at the first intron-exon junction.

4) The G at the released 5’ end of the intron folds back and forms an unusual 2’ to 5’ bond with the A in the branch point sequence.

5) This reaction produces a lariat structure.

6) Cleavage at the 3’ intron-exon junction and ligation of the two exons completes the removal of the intron.

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Model for intron removal by the spliceosome1) U1 and U2 snRNP bind to the 5’ splice

junction site and the branch point sequence, respectively.

2) Interactions with U4/U6 snRNP and U5 snRNP binds U1 and U2 together to form a loop.

3) U4 dissociates forming the active spliceosome.

4) The spliceosome complex cleave the intron from exon 1 at the 5’ splice junction. The free end binds to the A in the branch-point sequence (lariat).

5) The intron is excised by cleavage at the 3’ splice junction and exons 1 & 2 are ligated. SnRNPs are released.

RNA can be spliced in different ways to produce different species of mRNA in a process called alternative spicing.

Alternative RNA splicing can produce different forms of a protein from the same gene.

Four Patterns of Alternative Splicing

Blue=exon, yellow=intron

1) Optional exon - an exon may be included or excluded.

2) Optional intron - an intron may be included in the transcript or spliced out.

3) Mutually exclusive exons - an exon may be spliced out in one transcript and a different exon may be spliced out in another transcript.

4) Internal splice site - a weak splice site might exist within an intron to provide a secondary site of splicing.

Control of Alternative RNA Splicing:1) Alternative splicing can occur due to intron sequence

ambiguity. This may be due to ‘weak’ splice site sequences and therefore splicing choice occurs by chance.

2) Alternative splicing can also be directed by negative and positive control via proteins that bind to splice sequences and either repress or activate splicing at that site.

Sex Determination in the Fruitfly - Drosophila

The primary signal for determining whether a fly develops as a male or female is the X chromosome/autosome ratio: 1 (2 X-chromosomes/2 sets of autosomes) = female; 0.5 (1 X-chromosome/2 sets of autosomes) = male.

Three gene products are responsible for transmitting information about the sex ratio to cells.

The gene Sxl produces an initial alternate transcript that produces an active Sxl protein. This protein affects splicing of Sxl RNA and the RNA of tra and dsx.

In male Drosophila:

1) Sex-lethal (Sxl) and transformer (tra) constitutively spliced to form non-functional proteins.

2) As a result of non-functional tra protein, splicing of dsx RNA produces a protein that represses female differentiation genes.

In female Drosophila:

1) In females a transient Sxl transcript is produced that results in a functional Sxl protein. This protein binds to normal Sxl transcripts and produces an alternate splicing that produces functional protein.

2) Sxl protein blocks splicing of one exon in the tra transcript resulting in functional protein.

3) Tra protein activates a splice site in dsx that results in a protein that represses male differentiation genes.

RNA editing is a process in which information changes at the level of mRNA.

It is revealed by situations in which the coding sequence in an RNA differs from the sequence of DNA from which it was transcribed.

In mammalian cells there are cases in which a substitution occurs in an individual base in mRNA, causing a change in the sequence of the protein that is coded.

In trypanosome mitochondria, more widespread changes occur in transcripts when bases are added or deleted.

In mammals, the apo-B gene encodes two alternative forms of the apolipoprotein B: Apo-B100 (liver) and Apo-B48 (intestine).

In the intestine, the apo-B mRNA is edited so that a premature stop codon is produced (CAA -> UAA) leading to the synthesis of the shorter Apo-B48.

This editing is accomplished by a cytidine deaminase enzyme.

In trypansome editing is more extensive and involves complimentary base pairing by a “guide RNA”.

The editing is accomplished by an enzyme complex containing an endonuclease (cleavage), a terminal uridyltransferase (adding U), and an RNA ligase.

5’ 3’

5’ 3’endonuclease

5’ 3’U

Terminal uridyltransferase

5’ 3’U

RNA ligase

Steps of RNA editing in Trypanosome mitochondria:

1) Guide RNA complimentary base pairs with target RNA.

2) Endonuclease cleaves RNA at region of mispairing.

3) TUTase inserted uridine.

4) RNA ligase ligates substrate RNA.