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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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TbADAT2p localizes to both the nucleus and the cytoplasm. (A) Western blot with polyclonal antibodies specific for TbADAT2 (anti-TbADAT2). ‘Anti-enolase’ refers to polyclonal antibodies specific for enolase (a known cytoplasmic marker). (B) TbADAT2p was tagged with a TY epitope and expressed in procyclic T. brucei. ‘anti-TY’ refers to a western blot with antibodies specific for the epitope tag. ‘Anti-enolase’ refers to the enolase antibody used as a control as described above. ‘Total’, ‘cyto’ and ‘nuc’ refer to whole-cell, cytoplasmic and nuclear protein fractions, respectively. (C) Immunofluorescence experiments where cells transformed with the recombinant epitope-tagged TbADAT2 (TbADAT2-TY) (left panels) or cells transformed with TY alone (right panels) were stained with DAPI to determine the position of the nuclei (blue) or with fluorescent anti TY-antibodies (green). Two trypanosome cells are shown; in the first, TbADAT2-TY is detected within the nucleus (arrow); in the second, TbADAT2 is excluded from the nucleus. The location of the nucleus is marked with ‘n’; ‘k’ denotes the location of the kinetoplast (mitochondria).
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Figure 6: TbADAT2p localizes to both the nucleus and the cytoplasm. (A) Western blot with polyclonal antibodies specific for TbADAT2 (anti-TbADAT2). ‘Anti-enolase’ refers to polyclonal antibodies specific for enolase (a known cytoplasmic marker). (B) TbADAT2p was tagged with a TY epitope and expressed in procyclic T. brucei. ‘anti-TY’ refers to a western blot with antibodies specific for the epitope tag. ‘Anti-enolase’ refers to the enolase antibody used as a control as described above. ‘Total’, ‘cyto’ and ‘nuc’ refer to whole-cell, cytoplasmic and nuclear protein fractions, respectively. (C) Immunofluorescence experiments where cells transformed with the recombinant epitope-tagged TbADAT2 (TbADAT2-TY) (left panels) or cells transformed with TY alone (right panels) were stained with DAPI to determine the position of the nuclei (blue) or with fluorescent anti TY-antibodies (green). Two trypanosome cells are shown; in the first, TbADAT2-TY is detected within the nucleus (arrow); in the second, TbADAT2 is excluded from the nucleus. The location of the nucleus is marked with ‘n’; ‘k’ denotes the location of the kinetoplast (mitochondria).

Mentions: We have recently reported that the catalytic component of the A to I editing enzyme (ADAT2p) may play a role in both A to I and C to U editing in trypanosomatids, since down-regulation of TbADAT2p led to a concomitant decrease in both A to I and C to U editing levels in tRNAThrAGU (21). In light of our current results, we decided to also explore the sub-cellular localization of the ADAT2 protein. Protein fractions were isolated by similar methods as described in Materials and Methods section and then analyzed by western blot with antibodies specific for ADAT2. We observed a low but significant signal for this protein in both the cytoplasmic and nuclear fractions (Figure 6). However, due to the relative low titer of this antibody, we also performed similar analyses but with protein extracts from cells transformed with an epitope-tagged copy of ADAT2. Again we found a measurable amount of TbADAT2p localized to the nucleus (Figure 6). This finding was further supported by immunofluorescence assay that showed the co-localization of the ADAT2p signal with DAPI. Taken together, these observations support the view that ADAT2p's role in both editing events and its intracellular localization correlates with the distribution of C to U and A to I editing in these cells.Figure 6.


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)

TbADAT2p localizes to both the nucleus and the cytoplasm. (A) Western blot with polyclonal antibodies specific for TbADAT2 (anti-TbADAT2). ‘Anti-enolase’ refers to polyclonal antibodies specific for enolase (a known cytoplasmic marker). (B) TbADAT2p was tagged with a TY epitope and expressed in procyclic T. brucei. ‘anti-TY’ refers to a western blot with antibodies specific for the epitope tag. ‘Anti-enolase’ refers to the enolase antibody used as a control as described above. ‘Total’, ‘cyto’ and ‘nuc’ refer to whole-cell, cytoplasmic and nuclear protein fractions, respectively. (C) Immunofluorescence experiments where cells transformed with the recombinant epitope-tagged TbADAT2 (TbADAT2-TY) (left panels) or cells transformed with TY alone (right panels) were stained with DAPI to determine the position of the nuclei (blue) or with fluorescent anti TY-antibodies (green). Two trypanosome cells are shown; in the first, TbADAT2-TY is detected within the nucleus (arrow); in the second, TbADAT2 is excluded from the nucleus. The location of the nucleus is marked with ‘n’; ‘k’ denotes the location of the kinetoplast (mitochondria).
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Figure 6: TbADAT2p localizes to both the nucleus and the cytoplasm. (A) Western blot with polyclonal antibodies specific for TbADAT2 (anti-TbADAT2). ‘Anti-enolase’ refers to polyclonal antibodies specific for enolase (a known cytoplasmic marker). (B) TbADAT2p was tagged with a TY epitope and expressed in procyclic T. brucei. ‘anti-TY’ refers to a western blot with antibodies specific for the epitope tag. ‘Anti-enolase’ refers to the enolase antibody used as a control as described above. ‘Total’, ‘cyto’ and ‘nuc’ refer to whole-cell, cytoplasmic and nuclear protein fractions, respectively. (C) Immunofluorescence experiments where cells transformed with the recombinant epitope-tagged TbADAT2 (TbADAT2-TY) (left panels) or cells transformed with TY alone (right panels) were stained with DAPI to determine the position of the nuclei (blue) or with fluorescent anti TY-antibodies (green). Two trypanosome cells are shown; in the first, TbADAT2-TY is detected within the nucleus (arrow); in the second, TbADAT2 is excluded from the nucleus. The location of the nucleus is marked with ‘n’; ‘k’ denotes the location of the kinetoplast (mitochondria).
Mentions: We have recently reported that the catalytic component of the A to I editing enzyme (ADAT2p) may play a role in both A to I and C to U editing in trypanosomatids, since down-regulation of TbADAT2p led to a concomitant decrease in both A to I and C to U editing levels in tRNAThrAGU (21). In light of our current results, we decided to also explore the sub-cellular localization of the ADAT2 protein. Protein fractions were isolated by similar methods as described in Materials and Methods section and then analyzed by western blot with antibodies specific for ADAT2. We observed a low but significant signal for this protein in both the cytoplasmic and nuclear fractions (Figure 6). However, due to the relative low titer of this antibody, we also performed similar analyses but with protein extracts from cells transformed with an epitope-tagged copy of ADAT2. Again we found a measurable amount of TbADAT2p localized to the nucleus (Figure 6). This finding was further supported by immunofluorescence assay that showed the co-localization of the ADAT2p signal with DAPI. Taken together, these observations support the view that ADAT2p's role in both editing events and its intracellular localization correlates with the distribution of C to U and A to I editing in these cells.Figure 6.

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