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Two RNA recognition motif-containing proteins are plant mitochondrial editing factors.

Shi X, Hanson MR, Bentolila S - Nucleic Acids Res. (2015)

Bottom Line: The N-terminal RRM domain by itself provides the editing activity of ORRM3.In yeast-two hybrid assays, ORRM3 interacts with RIP1, ORRM2 and with itself.Identification of the effect of ORRM2 and ORRM3 on RNA editing reveals a previously undescribed role of RRM-containing proteins as mitochondrial RNA editing factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

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Residual extent in orrm3 mutant plants can be reduced by silencing ORRM2. (A) Juxtaposition of editing extent at two sites under the control of ORRM2 and ORRM3 in ORRM2-silenced orrm3 mutant plants and ORRM2 expression. A solid black line withsquares shows the relative ORRM2 expression in orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct and the controls. Bars refer to the editing extent at sites rps3 C1344 and nad6 leader C-73, measured by PPE assay. NOT, orrm3 mutant plants that were not inoculated with Agrobacteria; GFP-sil, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP silencing construct; sil-1 to sil-8, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct. (B) Editing extent in ORRM2-silenced orrm3 mutant plants correlates with the expression of ORRM2.
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Figure 8: Residual extent in orrm3 mutant plants can be reduced by silencing ORRM2. (A) Juxtaposition of editing extent at two sites under the control of ORRM2 and ORRM3 in ORRM2-silenced orrm3 mutant plants and ORRM2 expression. A solid black line withsquares shows the relative ORRM2 expression in orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct and the controls. Bars refer to the editing extent at sites rps3 C1344 and nad6 leader C-73, measured by PPE assay. NOT, orrm3 mutant plants that were not inoculated with Agrobacteria; GFP-sil, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP silencing construct; sil-1 to sil-8, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct. (B) Editing extent in ORRM2-silenced orrm3 mutant plants correlates with the expression of ORRM2.

Mentions: Given that ORRM2 interacts with ORRM3 in yeast, and that both factors control a rather large proportion of common target sites, we wondered whether they were also working synergistically in vivo. The lack of a T-DNA insertional mutant for ORRM2 prevented us from creating a double mutant, so we instead performed VIGS to transiently silence ORRM2 in orrm3 mutants. Two-week-old orrm3 mutant seedlings were inoculated with Agrobacterium strain carrying either the GFP silencing construct as a negative control or the ORRM2/GFP co-silencing construct (named sil-1 to sil-8 in Figure 8A). The relative expression level of ORRM2 in different plants was measured by quantitative RT-PCR, while the editing extent was examined by PPE assays. As shown in Figure 8, at sites rps3 C1344 and nad6 leader C-73, sites that are controlled by both ORRM2 and ORRM3, the variation of editing extent correlates well with the variation of ORRM2 expression level, especially for rps3 C1344 (Figure 8B). The expression of ORRM3 was not monitored in this experiment because we have shown previously that it is at an undetectable level in orrm3 mutant plants. The reduction of editing extent is more pronounced in plants with lower ORRM2 expression. For instance, the editing extent for both rps3 C1344 and nad6 leader C-73 is at its lowest for plants sil-1 and sil-8 which also exhibit the lowest (or close to the lowest for plant sil-1) level of ORRM2 expression (Figure 8A). The reduction of editing extent in plants sil-1 and sil-8 is significant compared to the uninoculated control plants for rps3 C1344 (P < 0.01), while for nad6 leader C-73 the reduction is significant when compared to both control plants, uninoculated (P < 0.01) and GFP-silenced (P < 0.05). The further reduction in the silenced plants result indicates that the residual editing extent of some sites in orrm3 mutants is dependent on the presence of ORRM2.


Two RNA recognition motif-containing proteins are plant mitochondrial editing factors.

Shi X, Hanson MR, Bentolila S - Nucleic Acids Res. (2015)

Residual extent in orrm3 mutant plants can be reduced by silencing ORRM2. (A) Juxtaposition of editing extent at two sites under the control of ORRM2 and ORRM3 in ORRM2-silenced orrm3 mutant plants and ORRM2 expression. A solid black line withsquares shows the relative ORRM2 expression in orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct and the controls. Bars refer to the editing extent at sites rps3 C1344 and nad6 leader C-73, measured by PPE assay. NOT, orrm3 mutant plants that were not inoculated with Agrobacteria; GFP-sil, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP silencing construct; sil-1 to sil-8, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct. (B) Editing extent in ORRM2-silenced orrm3 mutant plants correlates with the expression of ORRM2.
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Figure 8: Residual extent in orrm3 mutant plants can be reduced by silencing ORRM2. (A) Juxtaposition of editing extent at two sites under the control of ORRM2 and ORRM3 in ORRM2-silenced orrm3 mutant plants and ORRM2 expression. A solid black line withsquares shows the relative ORRM2 expression in orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct and the controls. Bars refer to the editing extent at sites rps3 C1344 and nad6 leader C-73, measured by PPE assay. NOT, orrm3 mutant plants that were not inoculated with Agrobacteria; GFP-sil, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP silencing construct; sil-1 to sil-8, orrm3 mutant plants inoculated with Agrobacteria harboring a GFP and ORRM2 co-silencing construct. (B) Editing extent in ORRM2-silenced orrm3 mutant plants correlates with the expression of ORRM2.
Mentions: Given that ORRM2 interacts with ORRM3 in yeast, and that both factors control a rather large proportion of common target sites, we wondered whether they were also working synergistically in vivo. The lack of a T-DNA insertional mutant for ORRM2 prevented us from creating a double mutant, so we instead performed VIGS to transiently silence ORRM2 in orrm3 mutants. Two-week-old orrm3 mutant seedlings were inoculated with Agrobacterium strain carrying either the GFP silencing construct as a negative control or the ORRM2/GFP co-silencing construct (named sil-1 to sil-8 in Figure 8A). The relative expression level of ORRM2 in different plants was measured by quantitative RT-PCR, while the editing extent was examined by PPE assays. As shown in Figure 8, at sites rps3 C1344 and nad6 leader C-73, sites that are controlled by both ORRM2 and ORRM3, the variation of editing extent correlates well with the variation of ORRM2 expression level, especially for rps3 C1344 (Figure 8B). The expression of ORRM3 was not monitored in this experiment because we have shown previously that it is at an undetectable level in orrm3 mutant plants. The reduction of editing extent is more pronounced in plants with lower ORRM2 expression. For instance, the editing extent for both rps3 C1344 and nad6 leader C-73 is at its lowest for plants sil-1 and sil-8 which also exhibit the lowest (or close to the lowest for plant sil-1) level of ORRM2 expression (Figure 8A). The reduction of editing extent in plants sil-1 and sil-8 is significant compared to the uninoculated control plants for rps3 C1344 (P < 0.01), while for nad6 leader C-73 the reduction is significant when compared to both control plants, uninoculated (P < 0.01) and GFP-silenced (P < 0.05). The further reduction in the silenced plants result indicates that the residual editing extent of some sites in orrm3 mutants is dependent on the presence of ORRM2.

Bottom Line: The N-terminal RRM domain by itself provides the editing activity of ORRM3.In yeast-two hybrid assays, ORRM3 interacts with RIP1, ORRM2 and with itself.Identification of the effect of ORRM2 and ORRM3 on RNA editing reveals a previously undescribed role of RRM-containing proteins as mitochondrial RNA editing factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

Show MeSH