Abstract
The interplay between mRNAs and proteins plays a crucial role in the regulation of cell morphology and physiology, and recent studies reveal that many proteins can serve as RNA‐binding proteins (RBPs) in addition to their previously characterized roles. By using the yeast as an organism model, we have tagged endogenously expressed mRNAs of interest with MS2 aptamer, followed by biochemical pull‐down and mass spectrometry analysis to reveal the identity of known proteins that act as RBPs in the trafficking of specific mRNAs. Here, we present two examples of novel RBPs essential for cellular physiological functions in yeast.
First, we have found that the COPI vesicle coat complex is necessary for the localization of mRNAs encoding mitochondrial proteins (mMPs) to mitochondria and for mitochondrial function. Inactivation of any one of the COPI proteins leads to reduced mMP binding to COPI itself, resulting in the dissociation of
mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI‐interaction site) led to altered mRNA localization and impaired cellular respiration (Zabezhinsky et al., Cell Rep (2016) 15:540‐549).
Second, we identified a histone 4 (Hhf1) as a protein that binds to STE2 mRNA, which encodes receptor for α‐factor and is involved in cell‐cell mating. Deletion of either one of the two paralogs of histone 4 led to a two‐fold reduction in mating efficiency, while the deletion of two specific histone acetyl transferases also had the same effect. By applying the CRISPR/Cas9 system in yeast we successfully mutated the endogenous acetylation sites of HHF1 and saw a marked reduction in mating efficiency. Furthermore, mutation of the acetylation sites in Hhf1 showed reduced binding to STE2 mRNA. Together, our approach shows that endogenous mRNA tagging followed by the pull‐down and analysis of bound protein can lead to surprising new insights into the functions of well‐known proteins.
First, we have found that the COPI vesicle coat complex is necessary for the localization of mRNAs encoding mitochondrial proteins (mMPs) to mitochondria and for mitochondrial function. Inactivation of any one of the COPI proteins leads to reduced mMP binding to COPI itself, resulting in the dissociation of
mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI‐interaction site) led to altered mRNA localization and impaired cellular respiration (Zabezhinsky et al., Cell Rep (2016) 15:540‐549).
Second, we identified a histone 4 (Hhf1) as a protein that binds to STE2 mRNA, which encodes receptor for α‐factor and is involved in cell‐cell mating. Deletion of either one of the two paralogs of histone 4 led to a two‐fold reduction in mating efficiency, while the deletion of two specific histone acetyl transferases also had the same effect. By applying the CRISPR/Cas9 system in yeast we successfully mutated the endogenous acetylation sites of HHF1 and saw a marked reduction in mating efficiency. Furthermore, mutation of the acetylation sites in Hhf1 showed reduced binding to STE2 mRNA. Together, our approach shows that endogenous mRNA tagging followed by the pull‐down and analysis of bound protein can lead to surprising new insights into the functions of well‐known proteins.
| Original language | English |
|---|---|
| Article number | B1079 |
| Pages (from-to) | 851-851 |
| Number of pages | 1 |
| Journal | Molecular Biology of the Cell |
| Volume | 27 |
| Issue number | 25 |
| DOIs | |
| State | Published - 15 Dec 2016 |
| Event | Annual Meeting of the American Society for Cell Biology - San Francisco, United States Duration: 3 Dec 2016 → 7 Dec 2016 |