Abstract
RNA editing is an essential mechanism by which target cytidine nucleotides are
modified to uridine in both mitochondrial and plastidial messenger RNA. There are 34
major editing sites in Arabidopsis thaliana chloroplasts that must be specifically
recognised by the editing machinery. PentatricoPeptide Repeat (PPR) proteins are
strongly implicated in this process.
The first aim of this thesis was to determine a complete catalogue of plastidial
editing sites and to measure the extent of editing in different tissue types. Variation in
editing extent points to the possibility of editing acting as a regulatory mechanism to
control the amount of correctly translated proteins in the cell.
The second aim of this thesis was to determine how editing sites are recognised.
Eighteen known PPR proteins recognise 30 out of 34 of the plastidial editing sites. By
aligning these protein sequences to their RNA targets, we have found that particular
residues in the PPR proteins correlate with target nucleotides and that all PPR editing
factors bind such that the C-terminal PPR motif aligns 4 nt upstream of the edited C.
These alignments were used to establish a code that allows us to align other PPR
proteins with their targets.
The third aim of this thesis was to use this code to discover new editing factors
by predicting their ability to recognise sites whose specificity factor was unknown.
Three previously unknown editing factors, AEF1, AEF2 and AEF3, that edit
atpF(12707), ndhB-1(97016) and psbE(64109) respectively, were identified and
verified experimentally.
The final aim of this thesis was to use the RNA recognition code to alter the
specificity of editing factors. The editing factors CLB19 and OTP82 were deliberately
modified, leading to predictable alterations in their specificity both in vivo and in vitro.
These results suggest that RNA editing factors could become innovative tools for
targeted RNA modification with exciting biotechnological applications if properly
implemented.
modified to uridine in both mitochondrial and plastidial messenger RNA. There are 34
major editing sites in Arabidopsis thaliana chloroplasts that must be specifically
recognised by the editing machinery. PentatricoPeptide Repeat (PPR) proteins are
strongly implicated in this process.
The first aim of this thesis was to determine a complete catalogue of plastidial
editing sites and to measure the extent of editing in different tissue types. Variation in
editing extent points to the possibility of editing acting as a regulatory mechanism to
control the amount of correctly translated proteins in the cell.
The second aim of this thesis was to determine how editing sites are recognised.
Eighteen known PPR proteins recognise 30 out of 34 of the plastidial editing sites. By
aligning these protein sequences to their RNA targets, we have found that particular
residues in the PPR proteins correlate with target nucleotides and that all PPR editing
factors bind such that the C-terminal PPR motif aligns 4 nt upstream of the edited C.
These alignments were used to establish a code that allows us to align other PPR
proteins with their targets.
The third aim of this thesis was to use this code to discover new editing factors
by predicting their ability to recognise sites whose specificity factor was unknown.
Three previously unknown editing factors, AEF1, AEF2 and AEF3, that edit
atpF(12707), ndhB-1(97016) and psbE(64109) respectively, were identified and
verified experimentally.
The final aim of this thesis was to use the RNA recognition code to alter the
specificity of editing factors. The editing factors CLB19 and OTP82 were deliberately
modified, leading to predictable alterations in their specificity both in vivo and in vitro.
These results suggest that RNA editing factors could become innovative tools for
targeted RNA modification with exciting biotechnological applications if properly
implemented.
| Original language | English |
|---|---|
| Qualification | Doctor of Philosophy |
| Publication status | Unpublished - 2015 |