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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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Related in: MedlinePlus

Stable expression of ORRM3 under a 35S promoter in orrm3–1 mutant plants complements the editing defects. orrm3–1, homozygous orrm3–1 mutant plants; orrm3–1 w/ 35S::ORRM3, orrm3–1 mutant plants transformed with a construct expressing ORRM3 driven by a 35S promoter.(A) Several transgenic mutant plants were assayed by PPE assay. E, edited band; U, unedited band; O, oligonucleotide. (B) Editing measured by bulk sequencing shows the complementation to be specific. Portion of electrophoretograms from RT-PCR bulk sequencing is shown. The editable C is shown in white letter in black background. Only the sites being affected in orrm3–1 mutant plants are complemented by stable expression of ORRM3 (bottom lane), whereas the sites not affected in orrm3–1 mutant are not complemented (upper lane).
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Figure 5: Stable expression of ORRM3 under a 35S promoter in orrm3–1 mutant plants complements the editing defects. orrm3–1, homozygous orrm3–1 mutant plants; orrm3–1 w/ 35S::ORRM3, orrm3–1 mutant plants transformed with a construct expressing ORRM3 driven by a 35S promoter.(A) Several transgenic mutant plants were assayed by PPE assay. E, edited band; U, unedited band; O, oligonucleotide. (B) Editing measured by bulk sequencing shows the complementation to be specific. Portion of electrophoretograms from RT-PCR bulk sequencing is shown. The editable C is shown in white letter in black background. Only the sites being affected in orrm3–1 mutant plants are complemented by stable expression of ORRM3 (bottom lane), whereas the sites not affected in orrm3–1 mutant are not complemented (upper lane).

Mentions: To further prove that a decrease or absence of ORRM3 expression is the actual cause of editing defects, we transformed homozygous orrm3 mutant plants with a construct expressing ORRM3 under the control of a 35S promoter. We performed genotyping to verify that plants surviving the Basta selection are homozygous for the orrm3 T-DNA insertion allele while carrying the ORRM3 transgene. All the transgenic plants exhibited a normal phenotype. The editing extents of several independent transgenic plants were analyzed by PPE and bulk sequencing assays. Compared to the homozygous mutants without the transgene, all the complemented plants expressing ORRM3 showed restoration of editing (Figure 5). The variation of editing extents in different complemented plants is likely a reflection of differential expression of transgenes due to positional effect. For instance, at site rps4 C77, the editing extent varies from 70 to 97% (Figure 5A), while the wild-type editing level at site rps4 C77 is about 70% (Figure 4A). Thus, rps4 C77 editing in most transgenic plants is restored to the wild-type level or even higher.


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

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

Stable expression of ORRM3 under a 35S promoter in orrm3–1 mutant plants complements the editing defects. orrm3–1, homozygous orrm3–1 mutant plants; orrm3–1 w/ 35S::ORRM3, orrm3–1 mutant plants transformed with a construct expressing ORRM3 driven by a 35S promoter.(A) Several transgenic mutant plants were assayed by PPE assay. E, edited band; U, unedited band; O, oligonucleotide. (B) Editing measured by bulk sequencing shows the complementation to be specific. Portion of electrophoretograms from RT-PCR bulk sequencing is shown. The editable C is shown in white letter in black background. Only the sites being affected in orrm3–1 mutant plants are complemented by stable expression of ORRM3 (bottom lane), whereas the sites not affected in orrm3–1 mutant are not complemented (upper lane).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402546&req=5

Figure 5: Stable expression of ORRM3 under a 35S promoter in orrm3–1 mutant plants complements the editing defects. orrm3–1, homozygous orrm3–1 mutant plants; orrm3–1 w/ 35S::ORRM3, orrm3–1 mutant plants transformed with a construct expressing ORRM3 driven by a 35S promoter.(A) Several transgenic mutant plants were assayed by PPE assay. E, edited band; U, unedited band; O, oligonucleotide. (B) Editing measured by bulk sequencing shows the complementation to be specific. Portion of electrophoretograms from RT-PCR bulk sequencing is shown. The editable C is shown in white letter in black background. Only the sites being affected in orrm3–1 mutant plants are complemented by stable expression of ORRM3 (bottom lane), whereas the sites not affected in orrm3–1 mutant are not complemented (upper lane).
Mentions: To further prove that a decrease or absence of ORRM3 expression is the actual cause of editing defects, we transformed homozygous orrm3 mutant plants with a construct expressing ORRM3 under the control of a 35S promoter. We performed genotyping to verify that plants surviving the Basta selection are homozygous for the orrm3 T-DNA insertion allele while carrying the ORRM3 transgene. All the transgenic plants exhibited a normal phenotype. The editing extents of several independent transgenic plants were analyzed by PPE and bulk sequencing assays. Compared to the homozygous mutants without the transgene, all the complemented plants expressing ORRM3 showed restoration of editing (Figure 5). The variation of editing extents in different complemented plants is likely a reflection of differential expression of transgenes due to positional effect. For instance, at site rps4 C77, the editing extent varies from 70 to 97% (Figure 5A), while the wild-type editing level at site rps4 C77 is about 70% (Figure 4A). Thus, rps4 C77 editing in most transgenic plants is restored to the wild-type level or even higher.

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
Related in: MedlinePlus