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 but not A to I editing is a nuclear event in tRNAThrAGU. (A) Total (T), nuclear (N) and cytoplasmic (C) RNA was purified as previously described (Materials and Methods section) and analyzed by northern blots. The same membrane was probed with either a spliced-leader RNA-specific probe, which is a mostly cytoplasmic RNA or with a U6 RNA-specific probe, a nucleus-specific marker, to assess the level of purity of each fraction. (B) The nuclear fraction from (A) was used for RT-PCR with tRNAThrAGU-specific primer. ‘RT+’ and ‘RT−’ refer to reactions performed either in the presence or absence of reverse transcriptase, where the RT− reaction serves as a control for DNA contamination. (C) The RT-PCR product from (B) was cloned into a plasmid vector and 65 independent clones sequenced. Only 1 out 66 clones analyzed contained the C to U editing at position 32 but none had the A to I editing at 34, these numbers were compared to our previous results with total RNA were 18 out of 30 clones were double edited. Similarly, a number of clones for the other two isoacceptors were analyzed and both were edited to significantly higher levels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: C to U but not A to I editing is a nuclear event in tRNAThrAGU. (A) Total (T), nuclear (N) and cytoplasmic (C) RNA was purified as previously described (Materials and Methods section) and analyzed by northern blots. The same membrane was probed with either a spliced-leader RNA-specific probe, which is a mostly cytoplasmic RNA or with a U6 RNA-specific probe, a nucleus-specific marker, to assess the level of purity of each fraction. (B) The nuclear fraction from (A) was used for RT-PCR with tRNAThrAGU-specific primer. ‘RT+’ and ‘RT−’ refer to reactions performed either in the presence or absence of reverse transcriptase, where the RT− reaction serves as a control for DNA contamination. (C) The RT-PCR product from (B) was cloned into a plasmid vector and 65 independent clones sequenced. Only 1 out 66 clones analyzed contained the C to U editing at position 32 but none had the A to I editing at 34, these numbers were compared to our previous results with total RNA were 18 out of 30 clones were double edited. Similarly, a number of clones for the other two isoacceptors were analyzed and both were edited to significantly higher levels.

Mentions: Although A to I editing of tRNA has been known for many years, little is known about the intracellular distribution of these editing events. In the case of A to I mRNA editing, it is well established that the editing enzyme localizes to the nucleus/nucleolus of mammalian cells (26). We decided to probe the intracellular distribution of the two editing events in trypanosomatids. We have previously developed purification methods that generate sub-cellular fractions with negligible cross-contamination (23). We have used similar methods to isolate total nuclear and cytoplasmic RNA fractions from T. brucei. These fractions were used in northern analysis to assess the relative purity of the fractions. RNAs were transferred to nitrocellulose membranes and hybridized to compartment-specific radioactive probes as described in the ‘Materials and Methods’ section. These probes were either specific for U6 snRNA (a nuclear marker) or spliced leader RNA (a nucleo-cytoplasmic marker which is predominant in the cytoplasm). As expected hybridization signals were observed which established that these fractions have little cross-contamination from other cellular compartments (Figure 4A). RNAs from these preparations were used for RT-PCR analysis as described earlier, where once again a number of independent clones were sequenced to assess editing levels (Figure 4B and C). All three isoacceptors showed C to U editing in the nuclear fractions with 17%, 14% and 1.5% edited for tRNAThrCGU, tRNAThrUGU and tRNAThrAGU, respectively. However, since every sub-cellular marker used in northern analysis to assess fraction purity corresponds to an RNA that is inevitably transcribed in the nucleus and transits to the cytoplasm, it could be argued that the observed values could be due to cytoplasmic contamination of our nuclear fractions. Notably, however, in the case of the tRNAThrAGU, no single clone out of a total of 66 analyzed contained the A to I editing event. Similar RT-PCR reactions were performed with tRNAValAAC, which also contains an adenosine at the first position of the anticodon and undergoes A to I editing. Again, no inosine-containing clone was observed with this particular tRNA, which in the cytoplasm is 97% edited from A to I (29 out of 30 clones) (data not shown). In addition, negligible A to I editing activity was detected when labeled tRNAThrAGU was incubated with nuclear protein fractions (data not shown). Taken together, this observation suggests that C to U editing of tRNAs occurs in the nucleus prior to export to the cytoplasm, while A to I editing is a cytoplasmic event.Figure 4.


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 but not A to I editing is a nuclear event in tRNAThrAGU. (A) Total (T), nuclear (N) and cytoplasmic (C) RNA was purified as previously described (Materials and Methods section) and analyzed by northern blots. The same membrane was probed with either a spliced-leader RNA-specific probe, which is a mostly cytoplasmic RNA or with a U6 RNA-specific probe, a nucleus-specific marker, to assess the level of purity of each fraction. (B) The nuclear fraction from (A) was used for RT-PCR with tRNAThrAGU-specific primer. ‘RT+’ and ‘RT−’ refer to reactions performed either in the presence or absence of reverse transcriptase, where the RT− reaction serves as a control for DNA contamination. (C) The RT-PCR product from (B) was cloned into a plasmid vector and 65 independent clones sequenced. Only 1 out 66 clones analyzed contained the C to U editing at position 32 but none had the A to I editing at 34, these numbers were compared to our previous results with total RNA were 18 out of 30 clones were double edited. Similarly, a number of clones for the other two isoacceptors were analyzed and both were edited to significantly higher levels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: C to U but not A to I editing is a nuclear event in tRNAThrAGU. (A) Total (T), nuclear (N) and cytoplasmic (C) RNA was purified as previously described (Materials and Methods section) and analyzed by northern blots. The same membrane was probed with either a spliced-leader RNA-specific probe, which is a mostly cytoplasmic RNA or with a U6 RNA-specific probe, a nucleus-specific marker, to assess the level of purity of each fraction. (B) The nuclear fraction from (A) was used for RT-PCR with tRNAThrAGU-specific primer. ‘RT+’ and ‘RT−’ refer to reactions performed either in the presence or absence of reverse transcriptase, where the RT− reaction serves as a control for DNA contamination. (C) The RT-PCR product from (B) was cloned into a plasmid vector and 65 independent clones sequenced. Only 1 out 66 clones analyzed contained the C to U editing at position 32 but none had the A to I editing at 34, these numbers were compared to our previous results with total RNA were 18 out of 30 clones were double edited. Similarly, a number of clones for the other two isoacceptors were analyzed and both were edited to significantly higher levels.
Mentions: Although A to I editing of tRNA has been known for many years, little is known about the intracellular distribution of these editing events. In the case of A to I mRNA editing, it is well established that the editing enzyme localizes to the nucleus/nucleolus of mammalian cells (26). We decided to probe the intracellular distribution of the two editing events in trypanosomatids. We have previously developed purification methods that generate sub-cellular fractions with negligible cross-contamination (23). We have used similar methods to isolate total nuclear and cytoplasmic RNA fractions from T. brucei. These fractions were used in northern analysis to assess the relative purity of the fractions. RNAs were transferred to nitrocellulose membranes and hybridized to compartment-specific radioactive probes as described in the ‘Materials and Methods’ section. These probes were either specific for U6 snRNA (a nuclear marker) or spliced leader RNA (a nucleo-cytoplasmic marker which is predominant in the cytoplasm). As expected hybridization signals were observed which established that these fractions have little cross-contamination from other cellular compartments (Figure 4A). RNAs from these preparations were used for RT-PCR analysis as described earlier, where once again a number of independent clones were sequenced to assess editing levels (Figure 4B and C). All three isoacceptors showed C to U editing in the nuclear fractions with 17%, 14% and 1.5% edited for tRNAThrCGU, tRNAThrUGU and tRNAThrAGU, respectively. However, since every sub-cellular marker used in northern analysis to assess fraction purity corresponds to an RNA that is inevitably transcribed in the nucleus and transits to the cytoplasm, it could be argued that the observed values could be due to cytoplasmic contamination of our nuclear fractions. Notably, however, in the case of the tRNAThrAGU, no single clone out of a total of 66 analyzed contained the A to I editing event. Similar RT-PCR reactions were performed with tRNAValAAC, which also contains an adenosine at the first position of the anticodon and undergoes A to I editing. Again, no inosine-containing clone was observed with this particular tRNA, which in the cytoplasm is 97% edited from A to I (29 out of 30 clones) (data not shown). In addition, negligible A to I editing activity was detected when labeled tRNAThrAGU was incubated with nuclear protein fractions (data not shown). Taken together, this observation suggests that C to U editing of tRNAs occurs in the nucleus prior to export to the cytoplasm, while A to I editing is a cytoplasmic event.Figure 4.

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