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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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All tRNAThr isoacceptors are substrate for aminoacylation in vivo. (A) Total RNA from T. brucei was extracted under acidic conditions and aminoacylation levels were correlated to editing levels by the oxopap assay as described. (B) Independent clones derived from the ‘+aa’ reaction (in A) were used in the OXOPAP assay (Materials and Methods section) and analyzed by sequencing. In all cases both edited and unedited species are substrates for aminoacylation in vivo. (C) Total RNA from the same fraction as above was also separated by acid denaturing polyacrylamide electrophoresis and probed with radioactive oligonucleotides specific for either tRNAThrAGU (top panel) or tRNAThrCGU/UGU. The probe used does not discriminate between the CGU and UGU isoacceptors. ‘−aa’ refers to a control reaction in which the RNA was deacylated by incubating under basic conditions prior to analyses. ‘+aa’ refers to the aminoacylated fractions purified and kept under acid pH.
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Figure 3: All tRNAThr isoacceptors are substrate for aminoacylation in vivo. (A) Total RNA from T. brucei was extracted under acidic conditions and aminoacylation levels were correlated to editing levels by the oxopap assay as described. (B) Independent clones derived from the ‘+aa’ reaction (in A) were used in the OXOPAP assay (Materials and Methods section) and analyzed by sequencing. In all cases both edited and unedited species are substrates for aminoacylation in vivo. (C) Total RNA from the same fraction as above was also separated by acid denaturing polyacrylamide electrophoresis and probed with radioactive oligonucleotides specific for either tRNAThrAGU (top panel) or tRNAThrCGU/UGU. The probe used does not discriminate between the CGU and UGU isoacceptors. ‘−aa’ refers to a control reaction in which the RNA was deacylated by incubating under basic conditions prior to analyses. ‘+aa’ refers to the aminoacylated fractions purified and kept under acid pH.

Mentions: Previously we showed that both the double-edited and unedited versions of tRNAThrAGU were actively aminoacylated. We also showed that although in vivo neither editing event played a major role as an aminoacylation determinant, the fact that both species are substrates for aminoacylation indicated that both were functional in translation. To examine the fate of the two edited isoacceptors, we performed similar experiments using our oxopap assay as previously described (18). Briefly, total RNA was isolated under acidic conditions and treated with sodium periodate which can oxidize free vicinal hydroxyls at the 3′ ends of RNA to form a dialdehyde, whereas tRNAs that bear an amino acid remain intact. Following oxidation, total tRNA was deacylated and incubated with ATP and poly(A) polymerase and used in a reverse transcription reaction with a tagged oligo-T primer specific for the poly(A) tail. Followed by PCR with a forward primer specific for the tRNA of interest and a reverse oligo complementary to the tag added during reverse transcription. The resulting products were cloned, transformed into E. coli and a number of independent clones analyzed by sequencing to assess the editing states of tRNAs that are aminoacylated in vivo. In this assay, only aminoacylated tRNAs are protected from periodate oxidation and can be amplified. A negative control of deacylated tRNA was used to show that only aminoacylated tRNAs are substrates for polyadenylation. (Figure 3A). We found that a majority of the clones sequenced were edited at C32 (65% and 68% for tRNAThrCGU and –UGU, respectively) (Figure 3B). Surprisingly these numbers are higher than those observed with total RNA. It is possible that during isolation the unedited tRNAs are preferentially deacylated, over the edited ones, which will skew the numbers from the oxidation-RT-PCR assay. To rule out this possibility, we also separated total RNA in an acid polyacrylamide gel for analysis of their aminoacylation extent by northern blots with radioactive probes specific for each tRNA. This experiment showed that a majority of the tRNA is aminoacylated in vivo and remained aminoacylated throughout the purification process (Figure 3C), as indicated by the shifted band observed during acid-gel electrophoresis as compared to a control RNA sample that was deacylated prior to electrophoresis. Alternatively, it is possible that the higher numbers may indicate a preference for the synthetase to charge the edited tRNAs, however we deem this possibility unlikely, given that aminoacylation experiments with the double-edited tRNA showed no such preference. Furthermore, should a preference exist, the acid gel northern analysis would have shown charging efficiencies commensurate with the 17–21% editing levels. Likely, the higher editing levels may be due to the presence of either oxidation-labile modifications (or some other modifications) that under normal RT-PCR conditions prevent amplification of all the tRNA species in a given sample and as such leads to a misrepresentation of the actual editing levels. Regardless, qualitatively these results show that, like in the case of the double-edited isoacceptor, these tRNAs are also functional in vivo in that they are efficiently utilized by the synthetase as a substrate.Figure 3.


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)

All tRNAThr isoacceptors are substrate for aminoacylation in vivo. (A) Total RNA from T. brucei was extracted under acidic conditions and aminoacylation levels were correlated to editing levels by the oxopap assay as described. (B) Independent clones derived from the ‘+aa’ reaction (in A) were used in the OXOPAP assay (Materials and Methods section) and analyzed by sequencing. In all cases both edited and unedited species are substrates for aminoacylation in vivo. (C) Total RNA from the same fraction as above was also separated by acid denaturing polyacrylamide electrophoresis and probed with radioactive oligonucleotides specific for either tRNAThrAGU (top panel) or tRNAThrCGU/UGU. The probe used does not discriminate between the CGU and UGU isoacceptors. ‘−aa’ refers to a control reaction in which the RNA was deacylated by incubating under basic conditions prior to analyses. ‘+aa’ refers to the aminoacylated fractions purified and kept under acid pH.
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Figure 3: All tRNAThr isoacceptors are substrate for aminoacylation in vivo. (A) Total RNA from T. brucei was extracted under acidic conditions and aminoacylation levels were correlated to editing levels by the oxopap assay as described. (B) Independent clones derived from the ‘+aa’ reaction (in A) were used in the OXOPAP assay (Materials and Methods section) and analyzed by sequencing. In all cases both edited and unedited species are substrates for aminoacylation in vivo. (C) Total RNA from the same fraction as above was also separated by acid denaturing polyacrylamide electrophoresis and probed with radioactive oligonucleotides specific for either tRNAThrAGU (top panel) or tRNAThrCGU/UGU. The probe used does not discriminate between the CGU and UGU isoacceptors. ‘−aa’ refers to a control reaction in which the RNA was deacylated by incubating under basic conditions prior to analyses. ‘+aa’ refers to the aminoacylated fractions purified and kept under acid pH.
Mentions: Previously we showed that both the double-edited and unedited versions of tRNAThrAGU were actively aminoacylated. We also showed that although in vivo neither editing event played a major role as an aminoacylation determinant, the fact that both species are substrates for aminoacylation indicated that both were functional in translation. To examine the fate of the two edited isoacceptors, we performed similar experiments using our oxopap assay as previously described (18). Briefly, total RNA was isolated under acidic conditions and treated with sodium periodate which can oxidize free vicinal hydroxyls at the 3′ ends of RNA to form a dialdehyde, whereas tRNAs that bear an amino acid remain intact. Following oxidation, total tRNA was deacylated and incubated with ATP and poly(A) polymerase and used in a reverse transcription reaction with a tagged oligo-T primer specific for the poly(A) tail. Followed by PCR with a forward primer specific for the tRNA of interest and a reverse oligo complementary to the tag added during reverse transcription. The resulting products were cloned, transformed into E. coli and a number of independent clones analyzed by sequencing to assess the editing states of tRNAs that are aminoacylated in vivo. In this assay, only aminoacylated tRNAs are protected from periodate oxidation and can be amplified. A negative control of deacylated tRNA was used to show that only aminoacylated tRNAs are substrates for polyadenylation. (Figure 3A). We found that a majority of the clones sequenced were edited at C32 (65% and 68% for tRNAThrCGU and –UGU, respectively) (Figure 3B). Surprisingly these numbers are higher than those observed with total RNA. It is possible that during isolation the unedited tRNAs are preferentially deacylated, over the edited ones, which will skew the numbers from the oxidation-RT-PCR assay. To rule out this possibility, we also separated total RNA in an acid polyacrylamide gel for analysis of their aminoacylation extent by northern blots with radioactive probes specific for each tRNA. This experiment showed that a majority of the tRNA is aminoacylated in vivo and remained aminoacylated throughout the purification process (Figure 3C), as indicated by the shifted band observed during acid-gel electrophoresis as compared to a control RNA sample that was deacylated prior to electrophoresis. Alternatively, it is possible that the higher numbers may indicate a preference for the synthetase to charge the edited tRNAs, however we deem this possibility unlikely, given that aminoacylation experiments with the double-edited tRNA showed no such preference. Furthermore, should a preference exist, the acid gel northern analysis would have shown charging efficiencies commensurate with the 17–21% editing levels. Likely, the higher editing levels may be due to the presence of either oxidation-labile modifications (or some other modifications) that under normal RT-PCR conditions prevent amplification of all the tRNA species in a given sample and as such leads to a misrepresentation of the actual editing levels. Regardless, qualitatively these results show that, like in the case of the double-edited isoacceptor, these tRNAs are also functional in vivo in that they are efficiently utilized by the synthetase as a substrate.Figure 3.

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