Thoreen, C. C. | Search bioRxiv

bioRxiv2024

Quantitative profiling of human translation initiation reveals regulatory elements that potently affect endogenous and therapeutically modified mRNAs

mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions and identify sequences that mediate 250-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1{Psi}) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissect mechanisms of human translation initiation and engineer more potent therapeutic mRNAs. HighlightsO_LIMeasurement of >30,000 human 5' UTRs reveals a 250-fold range of translation output C_LIO_LISystematic mutagenesis demonstrates the causality of short (3-6nt) regulatory elements C_LIO_LIN1-methylpseudouridine alters translation initiation in a sequence-specific manner C_LIO_LIOptimal modified 5' UTRs outperform those in the current class of mRNA vaccines C_LI

systems biology↗

bioRxiv2019

Archaeal ribosomal proteins possess nuclear localization signal-type motifs: implications for the origin of the cell nucleus

AO_SCPLOWBSTRACTC_SCPLOWEukaryotic cells are divided into the nucleus and the cytosol, and, to enter the nucleus, proteins typically possess short signal sequences, known as nuclear localization signals (NLSs). Although NLSs have long been considered as features unique to eukaryotic proteins, we show here that similar or identical protein segments are present in ribosomal proteins from the Archaea. Specifically, the ribosomal proteins uL3, uL15, uL18, and uS12 possess NLS-type motifs that are conserved across all major branches of the Archaea, including the most ancient groups Microarchaeota and Diapherotrites, pointing to the ancient origin of NLS-type motifs in the Archaea. Furthermore, by using fluorescence microscopy, we show that the archaeal NLS-type motifs can functionally substitute eukaryotic NLSs and direct the transport of ribosomal proteins into the nuclei of human cells. Collectively, these findings illustrate that the origin of NLSs preceded the origin of the cell nucleus, suggesting that the initial function of NLSs was not related to intracellular trafficking. Overall, our study reveals rare evolutionary intermediates among archaeal cells that can help elucidate the sequence of events that led to the origin of the eukaryotic cell.

evolutionary biology↗