In the process of mRNA translation into protein, transfer RNA (tRNA) is responsible for recognizing each codon on the mRNA and adding the corresponding amino acid to the polypeptide chain, which is then further folded and modified into protein. Recently, Professor Joshua Mendell's team at the University of Texas Southwestern Medical Center published a research paper titled "Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex to translating ribosomes" in the international top academic journal Science. Through cryo-electron microscopy and tRNA mutation experiments, the study found that the specific tRNA that decodes the arginine codon directly recruits the CCR4-NOT complex to the translating ribosome, initiates mRNA degradation, and thus promotes mRNA turnover. In contrast, some tRNAs have structural features that prevent the recruitment of the CCR4-NOT complex.
The study discovered a new role of tRNA in regulating mRNA degradation during translation - P-site tRNA-mediated mRNA decay, which expanded the known functions of tRNA and revealed a new mechanism for regulating mRNA stability in mammalian cells. This mechanism is crucial for regulating gene expression, especially mitochondrial-related mRNA. Therefore, this discovery may provide new treatments for mitochondrial genetic diseases, obesity, and mitochondrial-related diseases such as cancer.
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Many RNA-binding proteins and regulatory RNAs promote mRNA degradation by binding to specific messages and recruiting degradation factors, including the CCR4-NOT complex, which destabilizes mRNA by removing its poly(A) tail. Recent studies have shown that the CCR4-NOT complex can also be directly recruited to the ribosome when translation is inefficient. Specifically, when the ribosome encounters a codon with limited cognate tRNAs (non-optimal codons), it may pause in a configuration with empty A-site and E-site. This allows CNOT3, a subunit of the CCR4-NOT complex, to bind to the empty E-site, thereby promoting mRNA degradation and accelerating turnover.
High-throughput sequencing of mRNA imprints associated with CNOT3-bound ribosomes in human HEK293T cells revealed that the presence of slowly decoded codons in the A-site is not a strong signal for ribosome recruitment of CNOT3. In contrast, specific arginine codons (CGG, CGA, and AGG) in the P site were highly associated with CNOT3 recruitment, while other codons (including those specifying asparagine, lysine, isoleucine, tyrosine, phenylalanine, methionine, and threonine) were depleted from the P site of the CNOT3-bound ribosome.
Measurements of mRNA half-life and analysis of mRNA codon content showed that mRNAs encoding mitochondrial ribosomal proteins were highly enriched with CNOT3-associated arginine codons. Therefore, the CCR4-NOT complex is a powerful negative regulator of mitochondrial translation and quality. The research team said that because mitochondrial-associated mRNAs are most severely affected by this newly discovered mRNA degradation mechanism. In the future, it is expected that this degradation mechanism will be used to treat certain inherited mitochondrial diseases and other diseases in which mitochondria play a key role (such as obesity and cancer).
To investigate how P-site codon identity regulates CNOT3 recruitment, the research team performed cryo-EM analysis of CNOT3-bound ribosomes containing the CGG arginine codon. Combined with mutations in tRNA and CNOT3, the resulting high-resolution cryo-EM structure revealed a central role for P-site tRNA in CNOT3 recruitment. Specifically, CNOT3 enters the empty E-site and forms hydrogen-bonding interactions with the D-arm of the P-site tRNAArg,CCG. These interactions that promote CNOT3 recruitment depend on the presence of the rare U13:A22:A46 triplet in the arginine tRNAs that decode CGG, CGA, and AGG. In addition, tRNAs that decode codons missing from CNOT3-bound ribosomes often contain an extra nucleotide in the D-loop that sterically clashes with CNOT3, preventing its recruitment.
Figure 1.The P-site tRNA governs recruitment of the CCR4-NOT complex to translating ribosomes. (Zhu X, et al., 2024)
Overall, the study reveals that in addition to its classical role in mRNA decoding, tRNAs are also involved in ribosome recruitment of transcriptional regulators during translation, thereby promoting mRNA degradation and accelerating its turnover. The research team proposed P-site tRNA-mediated mRNA decay to describe this new mechanism of accelerated mRNA turnover.
Reference
Zhu X, et al. Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex to translating ribosomes. Science, 2024, 386(6724): eadq8587.