Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Haley R. Harrington; Matthew H. Zimmer; Laura M. Chamness; Veronica Nash; Wesley D. Penn; Thomas F. Miller; Suchetana Mukhopadhyay; Jonathan P. Schlebach

The apparent link between cotranslational folding and ribosomal frameshifting has implications for the mechanism of -1PRF in the SINV structural polyprotein. The portion of the transcript containing the EE and LL mutations is over 100 nucleotides upstream from the ribosomal slip site, and should therefore not perturb the stimulatory RNA structures that are currently believed to modulate -1PRF. 2, 22, 23 These mutations instead alter the portion of the nascent chain that falls just outside of the ribosomal exit tunnel during -1PRF, which suggests the nascent chain itself may stimulate frameshifting. Though it has yet to be implicated in ribosomal frameshifting, the cotranslational membrane integration and/ or folding of the nascent chain is known to generate tension on the ribosome. [41] [42] [43] [44] Furthermore, the C-terminal residue of TM2 is positioned 45 residues upstream of the slip site, which corresponds to a distance that should maximize the tension on the nascent chain at the moment the slip site occupies the ribosomal active site. 41, 42 Previous investigations have demonstrated that the force generated by the membrane integration of the nascent chain is sharply dependent upon this spacing. 41, 42 Therefore, to assess the potential role of this force in ribosomal frameshifting, we generated a set of SINV -1PRF reporter constructs (used in Fig. 4A ) containing a series of insertions and deletions that alter the distance between TM2 and the ribosomal slip site (see Supplemental Table 1 ). Reporter constructs were then expressed in HEK293T cells, and -1PRF reporter intensities were quantitatively compared at a uniform expression level by flow cytometry (Supplemental Fig. 5) . A comparison of reporter intensities reveals that -1PRF is maximized at the WT distance of 45 residues (Fig. 4B) . In all cases, deletions and insertions that change the distance between TM2 and the slip site significantly reduce the efficiency of -1PRF (Fig. 4B) . Moreover, the insertion of a 10-residue G/S linker decreases the intensity of the frameshift reporter by 76 ± 8% (n = 3), which suggests the membrane integration . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/790444 doi: bioRxiv preprint of TM2 is likely to be the primary driver of -1PRF within the SINV structural polyprotein.