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Sequencing RNAs that are biologically processed or degraded to less than ∼100 nt typically involves multistep, low-yield protocols with bias and information loss inherent to ligation and/or polynucleotide tailing. We recently introduced ordered two-template relay (OTTR), a method that captures obligatorily end-to-end sequences of input molecules and, in the same reverse transcription step, also appends 5' and 3' sequencing adapters of choice. OTTR has been thoroughly benchmarked for optimal production of microRNA, tRNA and tRNA fragments, and ribosome-protected mRNA footprint libraries. Here we sought to characterize, quantify, and ameliorate any remaining bias or imprecision in the end-to-end capture of RNA sequences. We introduce new metrics for the evaluation of sequence capture and use them to optimize reaction buffers, reverse transcriptase sequence, adapter oligonucleotides, and overall workflow. Modifications of the reverse transcriptase and adapter oligonucleotides increased the 3' and 5' end-precision of sequence capture and minimized overall library bias. Improvements in recombinant expression and purification of the truncated Bombyx mori R2 reverse transcriptase used in OTTR reduced nonproductive sequencing reads by minimizing bacterial nucleic acids that compete with low-input RNA molecules for cDNA synthesis, such that with miRNA input of 3 pg (<1 fmol), fewer than 10% of sequencing reads are bacterial nucleic acid contaminants. We also introduce a rapid, automation-compatible OTTR protocol that enables gel-free, length-agnostic enrichment of cDNA duplexes from unwanted adapter-only side products. Overall, this work informs considerations for unbiased end-to-end capture and annotation of RNAs independent of their sequence, structure, or posttranscriptional modifications., (© 2025 Ferguson et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The translation of messenger RNA (mRNA) into protein and the folding of the resulting protein into an active form are prerequisites for virtually every cellular process and represent the single largest investment of energy by cells. Ribosome profiling-based approaches have revolutionized our ability to monitor every step of protein synthesis in vivo, allowing one to measure the rate of protein synthesis across the proteome, annotate the protein coding capacity of genomes, monitor localized protein synthesis, and explore cotranslational folding and targeting. The rich and quantitative nature of ribosome profiling data provides an unprecedented opportunity to explore and model complex cellular processes. New analytical techniques and improved experimental protocols will provide a deeper understanding of the factors controlling translation speed and its impact on protein function and cell physiology as well as the role of ribosomal RNA and mRNA modifications in regulating translation., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5' end or subsequent scanning of the 5' UTR, but whether they perform unique or overlapping functions in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in budding yeast Saccharomyces cerevisiae by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A variant encoded by tif1-A79V (in a strain lacking the ortholog TIF2) yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5' UTR length and propensity for secondary structure, implicating Ded1 in scanning through structured 5' UTRs. Reporter assays confirmed that cap-distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs shows a heightened requirement for eIF4A, dependence on eIF4A is correlated with requirements for Ded1 and 5' UTR features characteristic of Ded1-dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5' UTRs, and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs., (© 2015 Sen et al.; Published by Cold Spring Harbor Laboratory Press.)

The recently developed ribosome profiling technique (Ribo-Seq) allows mapping of the locations of translating ribosomes on mRNAs with subcodon precision. When ribosome protected fragments (RPFs) are aligned to mRNA, a characteristic triplet periodicity pattern is revealed. We utilized the triplet periodicity of RPFs to develop a computational method for detecting transitions between reading frames that occur during programmed ribosomal frameshifting or in dual coding regions where the same nucleotide sequence codes for multiple proteins in different reading frames. Application of this method to ribosome profiling data obtained for human cells allowed us to detect several human genes where the same genomic segment is translated in more than one reading frame (from different transcripts as well as from the same mRNA) and revealed the translation of hitherto unpredicted coding open reading frames.

Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui(-) phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui(-) mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.