Ribosomal Frameshifting Selectively Modulates the Biosynthesis, Assembly, and Function of a Misfolded CFTR Variant

Abstract

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide influences the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the wild-type CFTR interactome, silent mutations that disrupt this RNA structure alter how ΔF508 CFTR interacts with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances both the expression and function of ΔF508 CFTR with no impact on wild-type. Finally, we show that disrupting this motif enhances the pharmacological rescue of ΔF508 by Trikafta, which implies ribosomal frameshifting antagonizes the effects of leading CF therapeutics. Together, our results reveal that ribosomal frameshifting selectively reduces the expression and assembly of a misfolded CFTR variant. These findings suggest cotranslational misfolding alters the processivity of translation and potentially the stability of the mRNA transcript through the dynamic modulation of ribosomal frameshifting.

CFTR Folding: From Structure and Proteostasis to Cystic Fibrosis Personalized Medicine

“…12,62 Translation, translocation, and co-and posttranslational folding occur at the ER membrane where CFTR is evaluated at multiple steps by membrane bound and cytosolic proteostasis factor. Translational dynamics at the ribosome impacts CFTR, with slower speeds increasing F508del folding 63,64 and ribosomal frameshifting sites modulating F508del function. 63 The Sec61 translocon, the ER membrane complex (EMC), 60,65 Hsp70 and Hsp90 chaperones play an integral role, with a myriad of cochaperones such as Hsp40/J proteins, nucleotide exchange factors (NEFs), and lectin binding chaperones also involved.…”

“…Translational dynamics at the ribosome impacts CFTR, with slower speeds increasing F508del folding 63,64 and ribosomal frameshifting sites modulating F508del function. 63 The Sec61 translocon, the ER membrane complex (EMC), 60,65 Hsp70 and Hsp90 chaperones play an integral role, with a myriad of cochaperones such as Hsp40/J proteins, nucleotide exchange factors (NEFs), and lectin binding chaperones also involved. F508del experiences a greater number and prolonged interactions with proteostasis factors compared to WT.…”

“…Numerous CFTR interactomics studies using proteomics methods (affinity-purification mass spectrometry , or proximity ligation (Bio-ID)), as well as two-hybrid high-throughput screening have revealed the proteostasis factors that interact with CFTR throughout its folding process. , Translation, translocation, and co- and posttranslational folding occur at the ER membrane where CFTR is evaluated at multiple steps by membrane bound and cytosolic proteostasis factor. Translational dynamics at the ribosome impacts CFTR, with slower speeds increasing F508del folding , and ribosomal frameshifting sites modulating F508del function . The Sec61 translocon, the ER membrane complex (EMC), , Hsp70 and Hsp90 chaperones play an integral role, with a myriad of cochaperones such as Hsp40/J proteins, nucleotide exchange factors (NEFs), and lectin binding chaperones also involved.…”