Abstract
The heterogeneity of ribosomes, characterized by structural variations, arises from differences in types, numbers, and/or post-translational modifications of participating ribosomal proteins (RPs), ribosomal RNAs (rRNAs) sequence variants plus post-transcriptional modifications, and additional molecules essential for forming a translational machinery. The ribosomal heterogeneity within an individual organism or a single cell leads to preferential translations of selected messenger RNA (mRNA) transcripts over others, especially in response to environmental cues. The role of ribosomal heterogeneity in SARS-CoV-2 coronavirus infection, propagation, related symptoms, or vaccine responses is not known, and a technique to examine these has not yet been developed. Tools to detect ribosomal heterogeneity or to profile translating mRNAs independently cannot identify unique or specialized ribosome(s) along with corresponding mRNA substrate(s). Concurrent characterizations of RPs and/or rRNAs with mRNA substrate from a single ribosome would be critical to decipher the putative role of ribosomal heterogeneity in the COVID-19 disease, caused by the SARS-CoV-2, which hijacks the host ribosome to preferentially translate its RNA genome. Such a protocol should be able to provide a high-throughput screening of clinical samples in a large population that would reach a statistical power for determining the impact of a specialized ribosome to specific characteristics of the disease. These characteristics may include host susceptibility, viral infectivity and transmissibility, severity of symptoms, antiviral treatment responses, and vaccine immunogenicity including its side effect and efficacy. In this study, several state-of-the-art techniques, in particular, chemical probing of ribosomal components or rRNA structures, proximity ligation to generate rRNA-mRNA chimeras for sequencing, nanopore gating of individual ribosomes, nanopore RNA sequencing and/or structural analyses, single-ribosome mass spectrometry, and microfluidic droplets for separating ribosomes or indexing rRNAs/mRNAs, are discussed. The key elements for further improvement and proper integration of the above techniques to potentially arrive at a high-throughput protocol for examining individual ribosomes and their mRNA substrates in a clinical setting are also presented.
Highlights
The heterogeneity of ribosomes, characterized by structural variations, arises from differences in types, numbers, and/or post-translational modifications of participating ribosomal proteins (RPs), ribosomal RNAs sequence variants plus post-transcriptional modifications, and additional molecules essential for forming a translational machinery
Interactions of viral proteins and RNAs with host ribosomal translational machinery have been extensively characterized in cultured cells by chemical and/or physical cross-linking strategies followed by mass spectrometry and/or RNA-sequencing
Ribosomal proteins and ribosomal RNAs (rRNAs) form complexes with other initiation and elongation partners, such as eukaryotic translation initiation factors (EIFs) and transfer RNAs (tRNAs), to perform translational activities
Summary
One direct evidence of ribosomal heterogeneity comes from ribosomopathy, caused by defective RPs and/or rRNAs. Further ribosomal profiling analysis revealed that ribosomes categorized by the presence or absence of tagged RPL10A or RPS25 display differential binding preference to distinct sets of transcripts [19]. Using such approach of tagging ribosomal proteins endogenously, several ribosome-associated proteins and preferential transcript substrates have been individually identified [20]. Replacement of maternal rRNAs with somatic rRNAs is observed during embryogenesis This shifts from the preferential binding of 18S expansion segment ES3 over ES6 onto maternally expressed mRNAs to binding of ES6 over ES3 onto somatically expressed mRNAs in silico assay [22]. The putative role of ribosomal heterogeneity in diseases or physiological protein biogenesis requires further investigation
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