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 L. Simpson K. Stuart

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 Maxi and mini circles are interlocked. 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

Some genes are very heavily edited! COXIII Cytochrome oxidase III From Trypanosoma brucei Lower case Us were inserted by editing. The deleted Ts (found in the DNA) are indicated in upper case. Fig. 16.15

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. Note also that G-U pairs are common in the dsRNA formed by the gRNA and the edited pre-mRNA. Editing is from 3’ to 5’ along an unedited RNA. 16.17,18

Editing Mechanism with the enzymes. TUTase, or terminal uridylyl transferase, adds U(s) to the 3’ end created by cleavage of the pre-mRNA from Fig. 16.20

Other Systems with RNA Editing Land plant (C  U) and Physarum (slime mold) mitochondria (nt insertions) Chloroplasts of angiosperms (C  U) Some nuclear genes in mammals Apolipoprotein B, C  U Glutamate receptor B, A  I (inosine) Hepatitus delta virus (A  I) Paramyxovirus (G insertions) Inosine is a purine, read as a G in mammalian translation.

Editing of Oenothera mitochondrial RNAs For a few mRNAs, e.g., the atp synthetase subunits, the corresponding protein was sequenced to verify the editing was functional. Determined by comparing sequences of cDNA copies of mt RNAs with the corresponding genomic gene.

Editing of Angiosperm Mt RNAs Most RNAs are edited Most events are C  U, but also U  C Preferential editing of coding regions, but introns and untranslated regions are also edited. Editing produces translatable RNAs, and restores conserved amino acids (i.e, functional proteins).

Possible mechanism for plant Mt editing: Deamination of cytosine (to uracil) by a cytidine deaminase NH2 O N N H20 = O = O N N Cytosine Uracil

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 Large nuclear gene Editing is C6666  U6666 in exon 26 of the 14 Kb mRNA 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 (Splicing precedes editing) All 3 kinds of RNA present, but just two forms of protein. Produced by Unedited mRNA Produced by Edited mRNA

Editing of Apolipoprotein B – The Editosome A cytidine deaminase activity is involved – apobec (apoB mRNA editing enzyme catalytic subunit) Another protein, ACF (apobec complementation factor) is also required 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 Inosine also acts as a signaling molecule. ADA deficiency is a metabolic disease.

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) converts A  I in 2 Glut Receptor B exons (changes the amino acids; I read as G during translation) recognizes secondary structure around site to be edited requires intron and exon sequences - acts on unspliced receptor pre-mRNA has dsRNA binding domains as well as a catalytic 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 Davidson, N. (2002) The Challenge of target-site specificity in C  U editing. J. Clinical Investigation 109: 291-294 Maas, S. et al. (2003) A-to-I RNA Editing:Recent News and Residual Mysteries*. J. Biol. Chemistry, 278, 1391-1394 Maas lab web site: www.lehigh.edu/~swm3/research.html dsRBD- dsRNA binding Z – Zn, DNA binding R-rich – Arg rich