Expression of therapeutically important proteins has benefited dramatically from the advent of chemically modified mRNAs that feature decreased lability and immunogenicity. This had a momentous effect on the rapid development of COVID-19 mRNA vaccines. Incorporation of the naturally occurring pseudouridine (Ψ) or N1-methyl-pseudouridine (N1mΨ) into in vitro transcribed mRNAs prevents the activation of unwanted immune responses by blocking eIF2α phosphorylation, which inhibits translation. Here, we report that Ψs in luciferase (Luc) mRNA exacerbate translation pausing in nuclease-untreated rabbit reticulocyte lysate (uRRL) and promote the formation of high-order-ribosome structures. The major deceleration of elongation occurs at the Ψ-rich nucleotides 1294–1326 of Ψ-Luc mRNA and results in premature termination of translation. The impairment of translation is mainly due to the shortage of membranous components. Supplementing uRRL with canine microsomal membranes (CMMs) relaxes the impediments to ribosome movement, resolves collided ribosomes, and greatly enhances full-size luciferase production. CMMs also strongly stimulated an extremely inefficient translation of N1mΨ-Luc mRNA in uRRL. Evidence is presented that translational pausing can promote membrane recruitment of polysomes with nascent polypeptides that lack a signal sequence. Our results highlight an underappreciated role of membrane binding to polysomes in the prevention of ribosome collision and premature release of nascent polypeptides.

The major mechanism of translation initiation in eukaryotes involves recognition of the cap structure at the 5′ end of the mRNA by the cap‐binding protein eIF4E. Internal ribosome entry sites (IRES) are specialised RNA sequences that are capable of recruiting ribosomes to an internal position of an mRNA in a cap‐independent manner. Many viruses have evolved this alternative pathway to initiate translation. Viral IRESs bind ribosomes by several mechanisms that require different sets of canonical initiation factors. In addition, IRES activity is modulated by IRES trans‐acting factors (ITAFs). Some cellular mRNAs are suggested to contain IRESs, as they function in cells under stress and other conditions when cap‐dependent initiation is inhibited. However, solid evidence for internal ribosome entry onto many of these mRNAs is lacking. Key Concepts In eukaryotes, the majority of mRNAs are translated by a 5′ end‐dependent mechanism, in which the mRNA 5′ cap structure recruits the translation machinery. Specific RNA elements, termed internal ribosome entry sites (IRESs), can direct ribosomes to internal positions of mRNAs to initiate translation. IRESs in viral RNA genomes differ significantly with respect to their structure and initiation factor requirements. In addition to some canonical initiation factors, IRES‐mediated translation may require ITAFs (IRES trans ‐acting factors). Many cellular mRNAs are suggested to possess IRESs, as they remain active under a number of pathophysiological stress conditions when cap‐dependent translation is inhibited. The authenticity of some cellular IRESs remains controversial as being not confirmed in recently developed stringent tests for IRES activity.

Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5′ untranslated regions (UTRs) or even lacking a 5′ UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.