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Our cells synthesize a range of proteins, each serving a unique function that, in numerous instances, requires them to be situated in specific areas of the cell where that function is essential. One technique that cells employ to ensure certain proteins are positioned correctly at the appropriate moment is localized translation, a mechanism that guarantees proteins are produced — or translated — close to their intended site of action. MIT professor of biology and member of the Whitehead Institute for Biomedical Research, Jonathan Weissman, along with his colleagues, has investigated localized translation to comprehend its influence on cellular functions and how cells can swiftly adapt to fluctuating environments.
Currently, Weissman, also a Howard Hughes Medical Institute Investigator, and postdoctoral researcher Jingchuan Luo have broadened our understanding of localized translation at mitochondria, the structures responsible for energy production within the cell. In an open-access publication released today in Cell, they introduce a novel tool, LOCL-TL, designed for in-depth examination of localized translation, and detail the insights gained about two categories of proteins that are locally translated at mitochondria.
The significance of localized translation at mitochondria stems from their unique origins. Mitochondria were once independent bacteria residing within our ancestral cells. Over time, these bacteria relinquished their independence and became incorporated into larger cells, migrating a majority of their genes into the nucleus of the larger cell. Cells have evolved methods to ensure that proteins required by mitochondria, encoded in genes within the larger cell’s genome, are transferred to the mitochondria. Although mitochondria preserve a few genes within their own genome, the synthesis of proteins from both the mitochondrial and nuclear genomes must be precisely coordinated to prevent the erroneous production of mitochondrial components. Localized translation may assist cells in managing the interaction between mitochondrial and nuclear protein synthesis — among other functions.
How to identify local protein synthesis
For a protein to be synthesized, the genetic code stored in DNA is transcribed into RNA, which is then translated by a ribosome, a cellular machinery that constructs a protein according to the RNA instructions. Weissman’s lab previously devised a technique to examine localized translation by tagging ribosomes near a structure of interest, subsequently capturing the tagged ribosomes in action and observing the proteins they are synthesizing. This technique, known as proximity-specific ribosome profiling, enables researchers to determine which proteins are being synthesized in particular areas of the cell. The obstacle that Luo encountered was modifying this method to solely capture ribosomes operating near mitochondria.
Ribosomes function rapidly, so a ribosome tagged while synthesizing protein at the mitochondria can rapidly transition to producing other proteins elsewhere in the cell within minutes. The only method by which researchers can ensure that the ribosomes they collect are still engaged in synthesizing proteins close to the mitochondria is if the procedure proceeds very swiftly.
Weissman and his team previously resolved this time-sensitivity challenge in yeast cells using a ribosome-tagging tool called BirA, which is activated by biotin presence. BirA is fused to the cellular structure of interest, tagging ribosomes it can contact — but only after activation. Researchers maintain biotin levels low until they are prepared to capture the ribosomes, thus limiting the duration of the tagging process. However, this strategy proves ineffective with mammalian cell mitochondria since they require biotin for normal function, making depletion impossible.
Luo and Weissman modified the existing tool to respond to blue light instead of biotin. The newly developed tool, LOV-BirA, is fused to the outer membrane of the mitochondrion. Cells are kept in darkness until the researchers are ready. Then, they illuminate the cells with blue light, activating LOV-BirA to tag ribosomes. After a brief interval, they swiftly extract the ribosomes. This method has demonstrated exceptional accuracy in capturing only ribosomes active at mitochondria.
The researchers then employed a technique originally devised by the Weissman lab to isolate RNA segments contained within the ribosomes. This allows them to ascertain how far along the ribosome is in the protein synthesis process at the moment of capture, which can reveal whether the entire protein is synthesized at the mitochondria or if it is partially produced elsewhere and completed at the mitochondria.
“One benefit of our tool is the detail it provides,” Luo explains. “Being able to identify which part of the protein is locally translated aids us in understanding more about how localized translation is regulated, which can subsequently help us comprehend its dysregulation in disease and to manipulate localized translation in future investigations.”
Two protein categories are synthesized at mitochondria
Utilizing these methods, the researchers discovered that approximately 20 percent of the genes required in mitochondria that reside in the main cellular genome are locally translated at mitochondria. These proteins can be categorized into two unique groups that possess differing evolutionary backgrounds and mechanisms for localized translation.
One group comprises relatively lengthy proteins, each containing over 400 amino acids or protein subsections. These proteins typically originate from bacteria — present in the ancestor of mitochondria — and are locally translated in both mammalian and yeast cells, indicating that their localized translation has been preserved over extensive evolutionary periods.
Similar to many mitochondrial proteins encoded in the nucleus, these proteins possess a mitochondrial targeting sequence (MTS), a molecular “ZIP code” that indicates to the cell where they should be delivered. The researchers found that most proteins with an MTS also feature a nearby inhibitory sequence that prevents transportation until they are fully synthesized. Conversely, this group of locally translated proteins lacks the inhibitory sequence, allowing them to reach the mitochondria during their production.
The synthesis of these longer proteins starts anywhere within the cell, and after roughly the first 250 amino acids are formed, they are transported to the mitochondria. While the remainder of the protein is created, it is simultaneously funneled into a pathway that ushers it inside the mitochondrion. This consumes the pathway for a considerable duration, restricting the import of other proteins, meaning cells can only afford to perform this simultaneous production and import for select proteins. The researchers theorize that these bacterial-origin proteins are prioritized as an ancient mechanism to ensure their precise production and placement within mitochondria.
The second group of locally translated proteins consists of brief proteins, each less than 200 amino acids in length. These proteins are evolutionarily more recent, and correspondingly, the researchers found that their localized translation mechanism is not shared by yeast. Their recruitment to mitochondria occurs at the RNA level. Two sequences within regulatory regions of each RNA molecule that do not code for the final protein instead encode for the cell’s machinery to attract the RNAs to the mitochondria.
The researchers sought out molecules that could be involved in this recruitment and identified the RNA binding protein AKAP1, which is present at the mitochondria. Upon eliminating AKAP1, the short proteins were synthesized indiscriminately throughout the cell. This provided an opportunity to gain insights about the implications of localized translation by observing what transpires in its absence. When the short proteins were not locally translated, this resulted in the loss of various mitochondrial proteins, including those required for oxidative phosphorylation, which is our cells’ primary energy production pathway.
In forthcoming research, Weissman and Luo will further explore how localized translation impacts mitochondrial function and dysfunction in disease. The researchers also plan to leverage LOCL-TL to investigate localized translation in other cellular phenomena, including embryonic development, neural plasticity, and various diseases.
“This method should be widely applicable to different cellular structures and types, opening numerous opportunities to comprehend how localized translation contributes to biological mechanisms,” Weissman remarks. “We are particularly keen on what we can uncover about its roles in ailments such as neurodegeneration, cardiovascular disorders, and cancers.”
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