RNA Editing

Definition: any process, other than splicing, that results in a change in the sequence of a RNA transcript such that it differs from the sequence of the DNA template

• Discovered in trypanosome mitochondria

• Also common in plant mitochondria• Also occurs in a few chloroplast genes of higher plants,

and at least a few nuclear genes in mammals

K. Stuart L. Simpson

Discovery of RNA Editing in Trypanosome Mitochondria

• Unusual Mitos. called Kinetoplasts• DNA:

– Maxicircles (22 kb in T. brucei), contains most of the genes

– Minicircles (1-3 kb), heterogenous

• Sequencing of genomic Mt DNA (Maxicircles) revealed apparent pseudogenes:– Full of Stop codons– Deletions of important amino acids

Kinetoplast DNA from a trypanosome visualized by EM

Fig. 16.13

Where were the real functional genes?

• Investigators generated cDNA clones to some of the kinetoplast mRNAs and sequenced them

• Sequences were partially complementary to pseudogenes on maxicircle DNA

cytochrome oxidase

subunit II

– the COXII DNA sequence above is missing 4 Us found in the mRNA

• Called this “Editing” because it produced functional mRNAs and proteins from pseudogenes

COXIIICytochrome oxidase III

From Trypanosoma brucei

Fig. 16.15

Lower case Us were inserted by editing.The deleted Ts (found in the DNA) are indicated in upper case.

Some genes are very heavily edited!

Editing Mechanism

• Post-transcriptional• Guide RNAs (gRNAs) direct editing

– gRNAs are small and complementary to portions of the edited mRNA

– Base-pairing of gRNA with unedited RNA gives mismatched regions, which are recognized by the editing machinery

– Machinery includes an Endonuclease, a Terminal UridylylTransferase (TUTase), and a RNA ligase

• Editing is directional, from 3’ to 5’

Guide RNAs Direct Editing in Trypanosomes.

Editing is from 3’ to 5’ along an unedited RNA.

16.17,18

TUTase, or terminal uridylyl transferase, adds U(s) to the 3’ end created by cleavage of the pre-mRNA

from Fig. 16.20

Editing Mechanism with the enzymes.

Other Systems with RNA Editing

1. Land plant (C U) and Physarum (slime mold) mitochondria (nt insertions)

2. Chloroplasts of angiosperms (C U)

3. Some nuclear genes in mammals – Apolipoprotein B, C U– Glutamate receptor B, A I (inosine)

4. Hepatitus delta virus (A I)

5. Paramyxovirus (G insertions)

Determined by comparing sequences of cDNA copies of mt RNAs with the corresponding genomic gene.

Editing of Oenothera mitochondrial RNAs

Editing of Angiosperm Mt RNAs

1. Most RNAs are edited

2. Most events are C U, but also U C

3. Preferential editing of coding regions, but introns and untranslated regions are also edited.

4. Editing produces translatable RNAs, and restores conserved amino acids (i.e, functional proteins).

N

N

= O

NH2

N

N

= O

O

Cytosine Uracil

H20

Possible mechanism for plant Mt editing: Deamination of cytosine (to uracil) by a cytidine deaminase

Plant mt RNA Editing Mechanism (cont.)

• Cytidine deaminases are known, and in fact one is involved in ApoB editing in mammals. Plant enzyme not identified yet.

• How are editing sites recognized?– No guide RNAs have yet been found in

angiosperm mitochondria.

Editing of Apolipoprotein B in Mammals

1. Large nuclear gene2. Editing is C6666 U6666 in exon 26 of the 14

Kb mRNA3. This creates a Stop codon, producing a

truncated form of the protein- both forms circulate in blood but have different functions- the long form is endocytosed via the

LDL receptor; the short form is not

Molecular Consequences of Editing ApoB pre-mRNA

Produced by Unedited mRNA Produced by Edited mRNA

(Splicing precedes editing)

Editing of Apolipoprotein B – The Editosome

1. A cytidine deaminase activity is involved– apobec (apoB mRNA editing enzyme catalytic subunit)

2. Another protein, ACF (apobec complementation factor) is also required

3. Both recognize sequences flanking the C to be edited

RNA editing in brain tissue: Adenosine to inosine

ADA

Adenosine Inosine

Inosine has long been known from purine metabolism

ADA deficiency is a metabolic disease.

Inosine also acts as a signaling molecule.

A to I Editing in RNA

• 1st case: Glutamate Receptor B

• I read as G during translation, R instead of Q

• Affects Ca2+

permeability, intracellular trafficking of receptor

Important Examples of A to I Editing in Mammals

Mechanism of A to I Editing

• dsRNA-dependent adenosine deaminase (ADAR)1. converts A I in 2 Glut Receptor B exons

(changes the amino acids; I read as G during translation)

2. recognizes secondary structure around site to be edited

3. requires intron and exon sequences - acts on unspliced receptor pre-mRNA

4. has dsRNA binding domains as well as acatalytic center similar to the cytosine deaminase

ADARs – adenosine deaminases that work on RNA

<- key for editing of GlutR

Other possible functions:- RNA modification (other

types)- RNAi- Chromatin remodeling

dsRBD- dsRNA bindingZ – Zn, DNA bindingR-rich – Arg rich