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1. Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon–anticodon pairing

   https://doi.org/10.7554/eLife.51898  

   Hoffer, Eric D; Hong, Samuel; Sunita, S; Maehigashi, Tatsuya; Gonzalez, Ruben L; Whitford, Paul C; Dunham, Christine M (October 2020, eLife)

   null (Ed.)

   Modifications in the tRNA anticodon loop, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguanosine modification at guanine nucleotide 37 (m 1 G37) located in the anticodon loop andimmediately adjacent to the anticodon nucleotides 34, 35, 36. The absence of m 1 G37 in tRNA Pro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNA Pro bound to either cognate or slippery codons to determine how the m 1 G37 modification prevents mRNA frameshifting. The structures reveal that certain codon–anticodon contexts and the lack of m 1 G37 destabilize interactions of tRNA Pro with the P site of the ribosome, causing large conformational changes typically only seen during EF-G-mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m 1 G37 stabilizes the interactions of tRNA Pro with the ribosome in the context of a slippery mRNA codon. 

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2. Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control

   https://doi.org/10.1093/nar/gkae285  

   Chen, Katharine_Y; Park, Heungwon; Subramaniam, Arvind_Rasi (April 2024, Nucleic Acids Research)

   Abstract Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level. 

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3. Mg+2 Ions Mediate the Interaction of Intrinsically Disordered Nascent Chains with the Ribosome

   Cavagnero, Silvia and (March 2022, The neuroscience chronicles)

   During the last few decades, the ribosome has been regarded primarily as a major cell player devoted to the catalysis of protein biosynthesis during translation [1-5]. It is therefore not surprising that several processes related to translation exploit the ribosome as a central hub. For instance, it is well-known that many events related to translational regulation are mediated by interactions between the ribosome and initiation, elongation or termination factors [6-9]. In addition, the ribosome is involved in mRNA-code recognition and proofreading [10-12] as well as in the control of translation rates via interactions with mRNA codons bearing high- and lowfrequency [13-15] and associated with variable tRNA abundance within the translation machinery [16-18]. Interestingly, the ribosome also assists de novo protein structure formation by minimizing cotranslational aggregation, thus increasing the yield of native-protein production [19,20]. The latter event, however, has not been shown to require -- or even involve -- direct interactions between the ribosome and the nascent protein chain. A notable exception is that of nascent chains bearing either N-terminal signal sequences or translational-arrest tags. These proteins are known to establish short- or long-term contacts with various regions of the ribosome during translation [21-25]. In summary, until recently very little knowledge has been available about direct contacts between the ribosome and nascent polypeptides and proteins that do not carry signal or arrest sequences. Studies based on fluorescence depolarization in the frequency domain [26] and NMR spectroscopy [27-30] provided interesting data that are consistent with, but do not unequivocally establish, the presence of these interactions. 

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4. Pervasive downstream RNA hairpins dynamically dictate start-codon selection

   https://doi.org/10.1038/s41586-023-06500-y  

   Xiang, Yezi; Huang, Wenze; Tan, Lianmei; Chen, Tianyuan; He, Yang; Irving, Patrick S.; Weeks, Kevin M.; Zhang, Qiangfeng Cliff; Dong, Xinnian (September 2023, Nature)

   Abstract Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites^1–4. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity inArabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming. 

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5. RiboA: a web application to identify ribosome A-site locations in ribosome profiling data

   https://doi.org/10.1186/s12859-021-04068-w  

   Shao, Danying; Ahmed, Nabeel; Soni, Nishant; O’Brien, Edward P. (December 2021, BMC Bioinformatics)

   null (Ed.)

   Abstract Background Translation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations. Results We present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms. Conclusions The Integer Programming method that RiboA utilizes is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at https://a-site.vmhost.psu.edu/ . The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license. 

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