Limits...
C to U editing at position 32 of the anticodon loop precedes tRNA 5' leader removal in trypanosomatids.

Gaston KW, Rubio MA, Spears JL, Pastar I, Papavasiliou FN, Alfonzo JD - Nucleic Acids Res. (2007)

Bottom Line: These involve 5' and 3' end trimming as well as the addition of a significant number of chemical modifications, including RNA editing.We also show that C to U editing is a nuclear event while A to I is cytoplasmic, where C to U editing at position 32 occurs in the precursor tRNA prior to 5' leader removal.Our data supports the view that C to U editing is more widespread than previously thought and is part of a stepwise process in the maturation of tRNAs in these organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Ohio State RNA Group, The Ohio State University, Columbus, Ohio 43210, USA.

ABSTRACT
In all organisms, precursor tRNAs are processed into mature functional units by post-transcriptional changes. These involve 5' and 3' end trimming as well as the addition of a significant number of chemical modifications, including RNA editing. The only known example of non-organellar C to U editing of tRNAs occurs in trypanosomatids. In this system, editing at position 32 of the anticodon loop of tRNA(Thr)(AGU) stimulates, but is not required for, the subsequent formation of inosine at position 34. In the present work, we expand the number of C to U edited tRNAs to include all the threonyl tRNA isoacceptors. Notably, the absence of a naturally encoded adenosine, at position 34, in two of these isoacceptors demonstrates that A to I is not required for C to U editing. We also show that C to U editing is a nuclear event while A to I is cytoplasmic, where C to U editing at position 32 occurs in the precursor tRNA prior to 5' leader removal. Our data supports the view that C to U editing is more widespread than previously thought and is part of a stepwise process in the maturation of tRNAs in these organisms.

Show MeSH

Related in: MedlinePlus

C to U editing precedes 5′ leader removal but occurs after 3′ maturation. (A) The nuclear RNA from above was the subject of RT-PCR analysis with the primers indicated, where primers 817 and 818 are specific for the 5′ leader and 3′ trailer, respectively. (B) The resulting RT-PCR reactions with all possible primer combinations were separated in a 3% agarose gel and stained with ethidium bromide. ‘+’ and ‘−’ refer to RT-PCR reactions with the same primer but performed in the presence and/or absence of reverse transcriptase, where the ‘−’ reaction is a mock control to check for DNA contamination in our RNA preparation. (C) The PCR products from (B) were cloned and sequenced. Out of 25 clones, 19 were derived from the leader containing RT-PCR reaction contained the C to U edit. No edited clones were observed in either the trailer-specific reaction (oligos 973/818) or the reaction specific for both precursors (oligos 817/818).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175311&req=5

Figure 5: C to U editing precedes 5′ leader removal but occurs after 3′ maturation. (A) The nuclear RNA from above was the subject of RT-PCR analysis with the primers indicated, where primers 817 and 818 are specific for the 5′ leader and 3′ trailer, respectively. (B) The resulting RT-PCR reactions with all possible primer combinations were separated in a 3% agarose gel and stained with ethidium bromide. ‘+’ and ‘−’ refer to RT-PCR reactions with the same primer but performed in the presence and/or absence of reverse transcriptase, where the ‘−’ reaction is a mock control to check for DNA contamination in our RNA preparation. (C) The PCR products from (B) were cloned and sequenced. Out of 25 clones, 19 were derived from the leader containing RT-PCR reaction contained the C to U edit. No edited clones were observed in either the trailer-specific reaction (oligos 973/818) or the reaction specific for both precursors (oligos 817/818).

Mentions: Marchfelder and co-workers showed that in plant mitochondria, C to U editing of tRNAs follow a specific sequence of events, where editing is required for 3′ processing of a 5′ matured tRNA. Therefore, in plant mitochondria C to U editing restores base pairing in the anticodon necessary for 3′ trailer removal. Our nuclear localization results have thus led us to explore the possibility that like in plant organelles nuclear tRNA editing also follows an orderly cascade. We generated oligonucleotide primers specific for either the 5′ or 3′ precursor tRNAs and used them in RT-PCR reactions (Figure 5). These reactions generated products of a size consistent with that of a pre-tRNA containing, either the 5′ leader, the 3′ trailer or both. These products were cloned into a plasmid vector individually analyzed, by cloning and sequencing a number of independent clones. We found that 76% (19 out of 25) of the 5′-precursor containing clones had undergone C to U editing at position 32, whereas none of the clones containing either a 3′ trailer or both the 5′ and 3′ extensions were edited (i.e. all have a C at position 32). Our observations reinforce the view that C to U editing is a nuclear, orderly process that occurs prior to 5′ end maturation and likely following 3′ end removal. This observation also suggests that in vivo the pre-tRNA is in fact the natural target of C to U editing.Figure 5.


C to U editing at position 32 of the anticodon loop precedes tRNA 5' leader removal in trypanosomatids.

Gaston KW, Rubio MA, Spears JL, Pastar I, Papavasiliou FN, Alfonzo JD - Nucleic Acids Res. (2007)

C to U editing precedes 5′ leader removal but occurs after 3′ maturation. (A) The nuclear RNA from above was the subject of RT-PCR analysis with the primers indicated, where primers 817 and 818 are specific for the 5′ leader and 3′ trailer, respectively. (B) The resulting RT-PCR reactions with all possible primer combinations were separated in a 3% agarose gel and stained with ethidium bromide. ‘+’ and ‘−’ refer to RT-PCR reactions with the same primer but performed in the presence and/or absence of reverse transcriptase, where the ‘−’ reaction is a mock control to check for DNA contamination in our RNA preparation. (C) The PCR products from (B) were cloned and sequenced. Out of 25 clones, 19 were derived from the leader containing RT-PCR reaction contained the C to U edit. No edited clones were observed in either the trailer-specific reaction (oligos 973/818) or the reaction specific for both precursors (oligos 817/818).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175311&req=5

Figure 5: C to U editing precedes 5′ leader removal but occurs after 3′ maturation. (A) The nuclear RNA from above was the subject of RT-PCR analysis with the primers indicated, where primers 817 and 818 are specific for the 5′ leader and 3′ trailer, respectively. (B) The resulting RT-PCR reactions with all possible primer combinations were separated in a 3% agarose gel and stained with ethidium bromide. ‘+’ and ‘−’ refer to RT-PCR reactions with the same primer but performed in the presence and/or absence of reverse transcriptase, where the ‘−’ reaction is a mock control to check for DNA contamination in our RNA preparation. (C) The PCR products from (B) were cloned and sequenced. Out of 25 clones, 19 were derived from the leader containing RT-PCR reaction contained the C to U edit. No edited clones were observed in either the trailer-specific reaction (oligos 973/818) or the reaction specific for both precursors (oligos 817/818).
Mentions: Marchfelder and co-workers showed that in plant mitochondria, C to U editing of tRNAs follow a specific sequence of events, where editing is required for 3′ processing of a 5′ matured tRNA. Therefore, in plant mitochondria C to U editing restores base pairing in the anticodon necessary for 3′ trailer removal. Our nuclear localization results have thus led us to explore the possibility that like in plant organelles nuclear tRNA editing also follows an orderly cascade. We generated oligonucleotide primers specific for either the 5′ or 3′ precursor tRNAs and used them in RT-PCR reactions (Figure 5). These reactions generated products of a size consistent with that of a pre-tRNA containing, either the 5′ leader, the 3′ trailer or both. These products were cloned into a plasmid vector individually analyzed, by cloning and sequencing a number of independent clones. We found that 76% (19 out of 25) of the 5′-precursor containing clones had undergone C to U editing at position 32, whereas none of the clones containing either a 3′ trailer or both the 5′ and 3′ extensions were edited (i.e. all have a C at position 32). Our observations reinforce the view that C to U editing is a nuclear, orderly process that occurs prior to 5′ end maturation and likely following 3′ end removal. This observation also suggests that in vivo the pre-tRNA is in fact the natural target of C to U editing.Figure 5.

Bottom Line: These involve 5' and 3' end trimming as well as the addition of a significant number of chemical modifications, including RNA editing.We also show that C to U editing is a nuclear event while A to I is cytoplasmic, where C to U editing at position 32 occurs in the precursor tRNA prior to 5' leader removal.Our data supports the view that C to U editing is more widespread than previously thought and is part of a stepwise process in the maturation of tRNAs in these organisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Ohio State RNA Group, The Ohio State University, Columbus, Ohio 43210, USA.

ABSTRACT
In all organisms, precursor tRNAs are processed into mature functional units by post-transcriptional changes. These involve 5' and 3' end trimming as well as the addition of a significant number of chemical modifications, including RNA editing. The only known example of non-organellar C to U editing of tRNAs occurs in trypanosomatids. In this system, editing at position 32 of the anticodon loop of tRNA(Thr)(AGU) stimulates, but is not required for, the subsequent formation of inosine at position 34. In the present work, we expand the number of C to U edited tRNAs to include all the threonyl tRNA isoacceptors. Notably, the absence of a naturally encoded adenosine, at position 34, in two of these isoacceptors demonstrates that A to I is not required for C to U editing. We also show that C to U editing is a nuclear event while A to I is cytoplasmic, where C to U editing at position 32 occurs in the precursor tRNA prior to 5' leader removal. Our data supports the view that C to U editing is more widespread than previously thought and is part of a stepwise process in the maturation of tRNAs in these organisms.

Show MeSH
Related in: MedlinePlus