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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.

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Related in: MedlinePlus

The T. brucei tRNAThrCGU and tRNAThrUGU undergo C to U editing in the anticodon loop. (A) RT-PCR with tRNAThrCGU and tRNAThrUGU-specific primers. The reactions were separated in a 3% agarose gel and stained with ethidium bromide. M, refers to a size marker lane. ‘DNA’ denotes a positive control lane in which a PCR reaction was performed with genomic DNA from T. brucei. ‘+’ 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. (B) A representative sequence of either a genomic DNA PCR product or one derived from the RT-PCR reaction. The arrow indicates the presence of the C to U editing event at position 32 of the anticodon loop, which is only present in the cDNA but not in the genomic DNA sequences. (C) A number of independent clones were sequenced from the RT-PCR products above, where 5 out 29 and 1 out of 29 clones were found edited for tRNAThrCGU in T. brucei (T.b.). Similar reactions as above yielded 19 out of 36 and 2 out of 33 clones edited in L. tarentolae (L.t.). No editing was detected in 30 clones derived from the genomic DNA PCR reaction.
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Figure 2: The T. brucei tRNAThrCGU and tRNAThrUGU undergo C to U editing in the anticodon loop. (A) RT-PCR with tRNAThrCGU and tRNAThrUGU-specific primers. The reactions were separated in a 3% agarose gel and stained with ethidium bromide. M, refers to a size marker lane. ‘DNA’ denotes a positive control lane in which a PCR reaction was performed with genomic DNA from T. brucei. ‘+’ 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. (B) A representative sequence of either a genomic DNA PCR product or one derived from the RT-PCR reaction. The arrow indicates the presence of the C to U editing event at position 32 of the anticodon loop, which is only present in the cDNA but not in the genomic DNA sequences. (C) A number of independent clones were sequenced from the RT-PCR products above, where 5 out 29 and 1 out of 29 clones were found edited for tRNAThrCGU in T. brucei (T.b.). Similar reactions as above yielded 19 out of 36 and 2 out of 33 clones edited in L. tarentolae (L.t.). No editing was detected in 30 clones derived from the genomic DNA PCR reaction.

Mentions: In all eukaryotes and most bacteria (but not archaea), tRNAs that are encoded with an adenosine at position 34 (the wobble nucleotide) are the subject of adenosine (A) to inosine (I) editing. Kinetoplastids are not an exception and in both T. brucei and Leishmania tarentolae all A34-containing tRNAs get edited, where I34 then permits decoding of the C-ending codons for the amino acids Ala, Arg, Ile, Leu, Pro, Ser, Thr and Val (Gaston and Alfonzo, unpublished data). Previously, we showed that tRNAThrAGU undergoes two distinct editing events in the anticodon loop, whereby every I34 containing tRNAThr also contains a C to U editing at position 32 in vivo (18). This observation raised the question of a possible interrelation between the two editing events, where either C to U is necessary for A to I formation or the reverse is true (i.e. A to I is necessary for C to U formation). Establishment of an efficient A to I editing assay led us to conclude that in vitro the presence of U32 (and no other nucleotide substitution at position 32) had a stimulatory effect, but it was not required, in the further formation of I34 (18). Still a standing question is whether the reverse is true, that A to I maybe required for C to U formation. The lack of an efficient in vitro C to U editing assay for tRNAs in any system (be it plants, marsupials or trypanosomatids) has thus far precluded answering some questions about editing and/or modification and their possible interrelation. To address the issue of editing site interrelation, we have used sequence comparative analysis coupled with an in vivo approach. We compared the sequence of the double-edited tRNA (tRNAThrAGU) to that of the two remaining tRNAThr isoacceptors (anticodon UGU and CGU) (Figure 1). All three tRNAs differ at a number of nucleotide positions in their backbone sequences (sequences not including the anticodon arm). Importantly, all three isoacceptors contain nearly identical anticodon loop sequences, including a C at position 32, the edited position in tRNAThrAGU, raising the possibility that the other two isoacceptors also undergo C to U editing in vivo. In addition, the L. tarentolae homologous isoacceptors have identical anticodon arm sequences to that of the T. brucei tRNAThr. We designed oligonucleotide primers specific for each of the two additional tRNAThr isoacceptors (Figure 1), whereas a 3′-specific oligomer was used to reverse transcribe tRNAThr from total T. brucei RNA. The resulting cDNA was then used as a template for PCR amplification with the same 3′ primer and a 5′-specific primer. Specific amplification products were obtained with these sets of primers when the reaction was performed in the presence of RT (Figure 2A), but was absent in ‘mock’ controls where the enzyme was omitted from the reactions. Indicating that the observed products are derived from reverse transcription of the RNA template and not from genomic DNA contamination. A product of identical size was obtained when both primers were used to amplify tRNAThr from total genomic DNA used as a positive control for amplification (Figure 2A). Similar results were obtained when L. tarentolae RNA and/or DNA was used in the RT-PCR and PCR reactions (data not shown). Both the cDNA-derived and the genomic DNA-derived products were then cloned into a plasmid vector, transformed into E. coli and a number of independent clones sequenced (Figure 2). We found that both tRNAThrCGU and -UGU undergo C to U editing at position 32 of the anticodon loop despite lacking an encoded A34 (Figure 2B). The observed editing (5 out of 29 clones, 17%) for the tRNAThrCGU and UGU (1 out of 30 clones, 3%) isoacceptors is lower than that observed for the double-edited tRNAThrAGU (18) (Figure 2C). Again similar numbers were obtained when the analogous products were sequenced from L. tarentolae sub-cellular fractions (Figure 2C). These results show that C to U editing of cytoplasmic tRNAs is more widespread than previously thought and is conserved among different trypanosomatid species. We also tested the possibility that other C32 containing tRNAs may undergo C to U editing at position 32, no editing was found in either tRNAArg or tRNAIle (data not shown). Notably these two tRNAs undergo A to I at position 34. Still our findings suggest that C to U editing may occur in other tRNAs in these organisms, perhaps at different positions, but this will remain an open question. The occurrence of C to U editing in tRNAs which lack an encoded A34 also rules out the possibility of inosine as a pre-requisite for C to U formation in these organisms.Figure 1.


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)

The T. brucei tRNAThrCGU and tRNAThrUGU undergo C to U editing in the anticodon loop. (A) RT-PCR with tRNAThrCGU and tRNAThrUGU-specific primers. The reactions were separated in a 3% agarose gel and stained with ethidium bromide. M, refers to a size marker lane. ‘DNA’ denotes a positive control lane in which a PCR reaction was performed with genomic DNA from T. brucei. ‘+’ 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. (B) A representative sequence of either a genomic DNA PCR product or one derived from the RT-PCR reaction. The arrow indicates the presence of the C to U editing event at position 32 of the anticodon loop, which is only present in the cDNA but not in the genomic DNA sequences. (C) A number of independent clones were sequenced from the RT-PCR products above, where 5 out 29 and 1 out of 29 clones were found edited for tRNAThrCGU in T. brucei (T.b.). Similar reactions as above yielded 19 out of 36 and 2 out of 33 clones edited in L. tarentolae (L.t.). No editing was detected in 30 clones derived from the genomic DNA PCR reaction.
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Figure 2: The T. brucei tRNAThrCGU and tRNAThrUGU undergo C to U editing in the anticodon loop. (A) RT-PCR with tRNAThrCGU and tRNAThrUGU-specific primers. The reactions were separated in a 3% agarose gel and stained with ethidium bromide. M, refers to a size marker lane. ‘DNA’ denotes a positive control lane in which a PCR reaction was performed with genomic DNA from T. brucei. ‘+’ 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. (B) A representative sequence of either a genomic DNA PCR product or one derived from the RT-PCR reaction. The arrow indicates the presence of the C to U editing event at position 32 of the anticodon loop, which is only present in the cDNA but not in the genomic DNA sequences. (C) A number of independent clones were sequenced from the RT-PCR products above, where 5 out 29 and 1 out of 29 clones were found edited for tRNAThrCGU in T. brucei (T.b.). Similar reactions as above yielded 19 out of 36 and 2 out of 33 clones edited in L. tarentolae (L.t.). No editing was detected in 30 clones derived from the genomic DNA PCR reaction.
Mentions: In all eukaryotes and most bacteria (but not archaea), tRNAs that are encoded with an adenosine at position 34 (the wobble nucleotide) are the subject of adenosine (A) to inosine (I) editing. Kinetoplastids are not an exception and in both T. brucei and Leishmania tarentolae all A34-containing tRNAs get edited, where I34 then permits decoding of the C-ending codons for the amino acids Ala, Arg, Ile, Leu, Pro, Ser, Thr and Val (Gaston and Alfonzo, unpublished data). Previously, we showed that tRNAThrAGU undergoes two distinct editing events in the anticodon loop, whereby every I34 containing tRNAThr also contains a C to U editing at position 32 in vivo (18). This observation raised the question of a possible interrelation between the two editing events, where either C to U is necessary for A to I formation or the reverse is true (i.e. A to I is necessary for C to U formation). Establishment of an efficient A to I editing assay led us to conclude that in vitro the presence of U32 (and no other nucleotide substitution at position 32) had a stimulatory effect, but it was not required, in the further formation of I34 (18). Still a standing question is whether the reverse is true, that A to I maybe required for C to U formation. The lack of an efficient in vitro C to U editing assay for tRNAs in any system (be it plants, marsupials or trypanosomatids) has thus far precluded answering some questions about editing and/or modification and their possible interrelation. To address the issue of editing site interrelation, we have used sequence comparative analysis coupled with an in vivo approach. We compared the sequence of the double-edited tRNA (tRNAThrAGU) to that of the two remaining tRNAThr isoacceptors (anticodon UGU and CGU) (Figure 1). All three tRNAs differ at a number of nucleotide positions in their backbone sequences (sequences not including the anticodon arm). Importantly, all three isoacceptors contain nearly identical anticodon loop sequences, including a C at position 32, the edited position in tRNAThrAGU, raising the possibility that the other two isoacceptors also undergo C to U editing in vivo. In addition, the L. tarentolae homologous isoacceptors have identical anticodon arm sequences to that of the T. brucei tRNAThr. We designed oligonucleotide primers specific for each of the two additional tRNAThr isoacceptors (Figure 1), whereas a 3′-specific oligomer was used to reverse transcribe tRNAThr from total T. brucei RNA. The resulting cDNA was then used as a template for PCR amplification with the same 3′ primer and a 5′-specific primer. Specific amplification products were obtained with these sets of primers when the reaction was performed in the presence of RT (Figure 2A), but was absent in ‘mock’ controls where the enzyme was omitted from the reactions. Indicating that the observed products are derived from reverse transcription of the RNA template and not from genomic DNA contamination. A product of identical size was obtained when both primers were used to amplify tRNAThr from total genomic DNA used as a positive control for amplification (Figure 2A). Similar results were obtained when L. tarentolae RNA and/or DNA was used in the RT-PCR and PCR reactions (data not shown). Both the cDNA-derived and the genomic DNA-derived products were then cloned into a plasmid vector, transformed into E. coli and a number of independent clones sequenced (Figure 2). We found that both tRNAThrCGU and -UGU undergo C to U editing at position 32 of the anticodon loop despite lacking an encoded A34 (Figure 2B). The observed editing (5 out of 29 clones, 17%) for the tRNAThrCGU and UGU (1 out of 30 clones, 3%) isoacceptors is lower than that observed for the double-edited tRNAThrAGU (18) (Figure 2C). Again similar numbers were obtained when the analogous products were sequenced from L. tarentolae sub-cellular fractions (Figure 2C). These results show that C to U editing of cytoplasmic tRNAs is more widespread than previously thought and is conserved among different trypanosomatid species. We also tested the possibility that other C32 containing tRNAs may undergo C to U editing at position 32, no editing was found in either tRNAArg or tRNAIle (data not shown). Notably these two tRNAs undergo A to I at position 34. Still our findings suggest that C to U editing may occur in other tRNAs in these organisms, perhaps at different positions, but this will remain an open question. The occurrence of C to U editing in tRNAs which lack an encoded A34 also rules out the possibility of inosine as a pre-requisite for C to U formation in these organisms.Figure 1.

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