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Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways.

Heininger AU, Hackert P, Andreou AZ, Boon KL, Memet I, Prior M, Clancy A, Schmidt B, Urlaub H, Schleiff E, Sloan KE, Deckers M, Lührmann R, Enderlein J, Klostermeier D, Rehling P, Bohnsack MT - RNA Biol (2016)

Bottom Line: We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase.Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis.Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43.

View Article: PubMed Central - PubMed

Affiliation: a Institute for Molecular Biology, Georg-August University , Goettingen , Germany.

ABSTRACT
A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

No MeSH data available.


Related in: MedlinePlus

G-patch protein overexpression leads to defects in ribosome biogenesis. (A) Scheme of the 35S pre-rRNA transcript, which contains the sequences of the mature 18S, 5.8S and 25S rRNAs. (B) Total RNA was isolated from yeast cells with or without overexpression of the individual G-patch proteins indicated, RNA was separated by denaturing agarose gel electrophoresis and analyzed by Northern blotting using probes for the detection of rRNA precursors (indicated on the left). The 35S pre-rRNA is marked by an arrowhead. (C) Levels of the 35S rRNA precursor transcript in three independent experiments were quantified, normalized to the scR1 RNA (loading control) and the wildtype, and are presented as mean +/− SEM.
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f0006: G-patch protein overexpression leads to defects in ribosome biogenesis. (A) Scheme of the 35S pre-rRNA transcript, which contains the sequences of the mature 18S, 5.8S and 25S rRNAs. (B) Total RNA was isolated from yeast cells with or without overexpression of the individual G-patch proteins indicated, RNA was separated by denaturing agarose gel electrophoresis and analyzed by Northern blotting using probes for the detection of rRNA precursors (indicated on the left). The 35S pre-rRNA is marked by an arrowhead. (C) Levels of the 35S rRNA precursor transcript in three independent experiments were quantified, normalized to the scR1 RNA (loading control) and the wildtype, and are presented as mean +/− SEM.

Mentions: Based on the competition of the cofactors and the relocalisation of Prp43 upon G-patch protein overexpression, we investigated whether the removal of Prp43 from target complexes leads to defects in pathways from which the RNA helicase is withdrawn. We analyzed the effects of G-patch protein overexpression on the processing of pre-rRNA intermediates (pre-rRNAs). Genetic depletion of Prp43 affects the biogenesis of both ribosomal subunits and impedes pre-rRNA processing, leading to a strong accumulation of the 35S pre-rRNA primary transcript (Fig. 6A).28,34 Here, we observed that overexpression of Spp2 or Spp382 did not significantly affect pre-rRNA processing (Fig. 6B-C), which is consistent with the absence or only minimal effects on Prp43 localization, respectively (Fig. 5). Conversely, overexpression of Sqs1 or Pxr1 caused accumulation of the 35S pre-rRNA transcript (Fig. 6B-C). The increase in 35S pre-rRNA levels resembles the effect of Prp43 depletion and is especially strong for overexpression of Sqs1. Sqs1 overexpression had also led to a major redistribution of Prp43 to the cytoplasm and therefore to a withdrawal of Prp43 from its interactions and functions in the nucleolus (Fig. 5). Although Prp43 is still localized in the nucleolus upon overexpression of Pxr1, a significant accumulation of the 35S pre-rRNA was also observed, suggesting that Prp43 is sequestered by the additional Pxr1 and therefore not able to fulfil its functions in the early steps of ribosome biogenesis. An increase in the level of the 35S pre-rRNA was also observed upon Cmg1 overexpression, but this effect is much milder, likely because a significant fraction of Cmg1 is sequestered in mitochondria and not accessible to Prp43. Taken together, our findings imply that Prp43-interacting proteins compete for the binding and recruitment of the RNA helicase and that the distribution of Prp43 can be regulated by changes in the availability of interaction partners.Figure 6.


Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways.

Heininger AU, Hackert P, Andreou AZ, Boon KL, Memet I, Prior M, Clancy A, Schmidt B, Urlaub H, Schleiff E, Sloan KE, Deckers M, Lührmann R, Enderlein J, Klostermeier D, Rehling P, Bohnsack MT - RNA Biol (2016)

G-patch protein overexpression leads to defects in ribosome biogenesis. (A) Scheme of the 35S pre-rRNA transcript, which contains the sequences of the mature 18S, 5.8S and 25S rRNAs. (B) Total RNA was isolated from yeast cells with or without overexpression of the individual G-patch proteins indicated, RNA was separated by denaturing agarose gel electrophoresis and analyzed by Northern blotting using probes for the detection of rRNA precursors (indicated on the left). The 35S pre-rRNA is marked by an arrowhead. (C) Levels of the 35S rRNA precursor transcript in three independent experiments were quantified, normalized to the scR1 RNA (loading control) and the wildtype, and are presented as mean +/− SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4829300&req=5

f0006: G-patch protein overexpression leads to defects in ribosome biogenesis. (A) Scheme of the 35S pre-rRNA transcript, which contains the sequences of the mature 18S, 5.8S and 25S rRNAs. (B) Total RNA was isolated from yeast cells with or without overexpression of the individual G-patch proteins indicated, RNA was separated by denaturing agarose gel electrophoresis and analyzed by Northern blotting using probes for the detection of rRNA precursors (indicated on the left). The 35S pre-rRNA is marked by an arrowhead. (C) Levels of the 35S rRNA precursor transcript in three independent experiments were quantified, normalized to the scR1 RNA (loading control) and the wildtype, and are presented as mean +/− SEM.
Mentions: Based on the competition of the cofactors and the relocalisation of Prp43 upon G-patch protein overexpression, we investigated whether the removal of Prp43 from target complexes leads to defects in pathways from which the RNA helicase is withdrawn. We analyzed the effects of G-patch protein overexpression on the processing of pre-rRNA intermediates (pre-rRNAs). Genetic depletion of Prp43 affects the biogenesis of both ribosomal subunits and impedes pre-rRNA processing, leading to a strong accumulation of the 35S pre-rRNA primary transcript (Fig. 6A).28,34 Here, we observed that overexpression of Spp2 or Spp382 did not significantly affect pre-rRNA processing (Fig. 6B-C), which is consistent with the absence or only minimal effects on Prp43 localization, respectively (Fig. 5). Conversely, overexpression of Sqs1 or Pxr1 caused accumulation of the 35S pre-rRNA transcript (Fig. 6B-C). The increase in 35S pre-rRNA levels resembles the effect of Prp43 depletion and is especially strong for overexpression of Sqs1. Sqs1 overexpression had also led to a major redistribution of Prp43 to the cytoplasm and therefore to a withdrawal of Prp43 from its interactions and functions in the nucleolus (Fig. 5). Although Prp43 is still localized in the nucleolus upon overexpression of Pxr1, a significant accumulation of the 35S pre-rRNA was also observed, suggesting that Prp43 is sequestered by the additional Pxr1 and therefore not able to fulfil its functions in the early steps of ribosome biogenesis. An increase in the level of the 35S pre-rRNA was also observed upon Cmg1 overexpression, but this effect is much milder, likely because a significant fraction of Cmg1 is sequestered in mitochondria and not accessible to Prp43. Taken together, our findings imply that Prp43-interacting proteins compete for the binding and recruitment of the RNA helicase and that the distribution of Prp43 can be regulated by changes in the availability of interaction partners.Figure 6.

Bottom Line: We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase.Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis.Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43.

View Article: PubMed Central - PubMed

Affiliation: a Institute for Molecular Biology, Georg-August University , Goettingen , Germany.

ABSTRACT
A rapidly increasing number of RNA helicases are implicated in several distinct cellular processes, however, the modes of regulation of multifunctional RNA helicases and their recruitment to different target complexes have remained unknown. Here, we show that the distribution of the multifunctional DEAH-box RNA helicase Prp43 between its diverse cellular functions can be regulated by the interplay of its G-patch protein cofactors. We identify the orphan G-patch protein Cmg1 (YLR271W) as a novel cofactor of Prp43 and show that it stimulates the RNA binding and ATPase activity of the helicase. Interestingly, Cmg1 localizes to the cytoplasm and to the intermembrane space of mitochondria and its overexpression promotes apoptosis. Furthermore, our data reveal that different G-patch protein cofactors compete for interaction with Prp43. Changes in the expression levels of Prp43-interacting G-patch proteins modulate the cellular localization of Prp43 and G-patch protein overexpression causes accumulation of the helicase in the cytoplasm or nucleoplasm. Overexpression of several G-patch proteins also leads to defects in ribosome biogenesis that are consistent with withdrawal of the helicase from this pathway. Together, these findings suggest that the availability of cofactors and the sequestering of the helicase are means to regulate the activity of multifunctional RNA helicases and their distribution between different cellular processes.

No MeSH data available.


Related in: MedlinePlus