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Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA.

Brown TA, Tkachuk AN, Clayton DA - PLoS ONE (2015)

Bottom Line: Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid.The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences.TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.

ABSTRACT
Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

No MeSH data available.


Relative levels of TFAM-immunoprecipitated and total cellular mitochondrial tRNAs determined by RT-PCR.(A) Ranked relative levels of mitochondrial tRNAs purified with TFAM in RNA immunoprecipitations. (B) Relative detectable levels of mitochondrial tRNAs obtained from 1x106 3T3sw cells.
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pone.0142436.g003: Relative levels of TFAM-immunoprecipitated and total cellular mitochondrial tRNAs determined by RT-PCR.(A) Ranked relative levels of mitochondrial tRNAs purified with TFAM in RNA immunoprecipitations. (B) Relative detectable levels of mitochondrial tRNAs obtained from 1x106 3T3sw cells.

Mentions: Since the EMSA binding data indicated that TFAM binds to complex RNA structures, we sought to identify TFAM-bound mitochondrial RNAs by using RNA immunoprecipitation (RIP) [20]. We used quantitative RT-PCR to identify RNAs that co-purified with TFAM using formaldehyde cross-linked cells. Our data indicate that TFAM does not appreciably immunoprecipitate mitochondrial mRNAs. We failed to find RNAs corresponding to 12S rRNA, ND1, ND2, CO1, COII, COIII ATP8/6, COIII, ND3, ND4, ND5, ND6 and Cytb in either the sense or antisense direction. However, we were able to easily detect 20 of the 22 mitochondrial tRNAs. We were unable to design PCR primers suitable for the detection of mt-tRNA Asp and mt-tRNA Ile; therefore, these tRNAs are missing from our data set. A small amount of 16S rRNA was found in the immunoprecipitations at a level below that of all but mt-tRNA LeuUUR, which was below detection. The relative ability to detect tRNAs purified by TFAM RIP are ranked and displayed in Fig 3A. As shown, mitochondrial tRNA levels are detected over a broad range. We first sought to determine if this variation simply reflected the relative steady state levels of each tRNA within cells. We prepared total RNA from cells and performed quantitative RT-PCR to determine relative levels of mitochondrial tRNAs, and found little correlation between the amount of tRNA present in the cell and that which we purified by TFAM RIP (Fig 3B). The mt-tRNA LeuUUR was undetectable in the TFAM immunoprecipitates, although our cellular assays indicated that this tRNA was more available than 9 of the other 19 tRNAs detected. Thus, although total cellular tRNA abundance may partially influence the amount that is bound to TFAM, it is not the primary factor. We also looked for TFAM binding correlations related to tRNA structural features without success. The determinants influencing the ability of TFAM to bind to a particular tRNA or our ability to detect those tRNAs is therefore not yet clear.


Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA.

Brown TA, Tkachuk AN, Clayton DA - PLoS ONE (2015)

Relative levels of TFAM-immunoprecipitated and total cellular mitochondrial tRNAs determined by RT-PCR.(A) Ranked relative levels of mitochondrial tRNAs purified with TFAM in RNA immunoprecipitations. (B) Relative detectable levels of mitochondrial tRNAs obtained from 1x106 3T3sw cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4636309&req=5

pone.0142436.g003: Relative levels of TFAM-immunoprecipitated and total cellular mitochondrial tRNAs determined by RT-PCR.(A) Ranked relative levels of mitochondrial tRNAs purified with TFAM in RNA immunoprecipitations. (B) Relative detectable levels of mitochondrial tRNAs obtained from 1x106 3T3sw cells.
Mentions: Since the EMSA binding data indicated that TFAM binds to complex RNA structures, we sought to identify TFAM-bound mitochondrial RNAs by using RNA immunoprecipitation (RIP) [20]. We used quantitative RT-PCR to identify RNAs that co-purified with TFAM using formaldehyde cross-linked cells. Our data indicate that TFAM does not appreciably immunoprecipitate mitochondrial mRNAs. We failed to find RNAs corresponding to 12S rRNA, ND1, ND2, CO1, COII, COIII ATP8/6, COIII, ND3, ND4, ND5, ND6 and Cytb in either the sense or antisense direction. However, we were able to easily detect 20 of the 22 mitochondrial tRNAs. We were unable to design PCR primers suitable for the detection of mt-tRNA Asp and mt-tRNA Ile; therefore, these tRNAs are missing from our data set. A small amount of 16S rRNA was found in the immunoprecipitations at a level below that of all but mt-tRNA LeuUUR, which was below detection. The relative ability to detect tRNAs purified by TFAM RIP are ranked and displayed in Fig 3A. As shown, mitochondrial tRNA levels are detected over a broad range. We first sought to determine if this variation simply reflected the relative steady state levels of each tRNA within cells. We prepared total RNA from cells and performed quantitative RT-PCR to determine relative levels of mitochondrial tRNAs, and found little correlation between the amount of tRNA present in the cell and that which we purified by TFAM RIP (Fig 3B). The mt-tRNA LeuUUR was undetectable in the TFAM immunoprecipitates, although our cellular assays indicated that this tRNA was more available than 9 of the other 19 tRNAs detected. Thus, although total cellular tRNA abundance may partially influence the amount that is bound to TFAM, it is not the primary factor. We also looked for TFAM binding correlations related to tRNA structural features without success. The determinants influencing the ability of TFAM to bind to a particular tRNA or our ability to detect those tRNAs is therefore not yet clear.

Bottom Line: Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid.The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences.TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.

ABSTRACT
Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

No MeSH data available.