Nascent Peptide-dependent Translation Arrest Leads to Not4p-mediated Protein Degradation by the Proteasome

Abstract

The potentially deleterious effects of aberrant mRNA lacking a termination codon (nonstop mRNA) are ameliorated by translation arrest, proteasome-mediated protein destabilization, and rapid mRNA degradation. Because polylysine synthesis via translation of the poly(A) mRNA tail leads to translation arrest and protein degradation by the proteasome, we examined the effects of other amino acid sequences. Insertion of 12 consecutive basic amino acids between GFP and HIS3 reporter genes, but not a stemloop structure, resulted in degradation of the truncated green fluorescent protein (GFP) products by the proteasome. Translation arrest products derived from GFP-R12-FLAG-HIS3 or GFP-K12-FLAG-HIS3 mRNA were detected in a not4⌬ mutant, and MG132 treatment did not affect the levels of the truncated arrest products. Deletion of other components of the Ccr4-Not complex did not increase the levels of the translation arrest products or reporter mRNAs. A L35A substitution in the Not4p RING finger domain, which disrupted its interaction with the Ubc4/Ubc5 E2 enzyme and its activity as an ubiquitin-protein ligase, also abrogated the degradation of arrest products. These results suggest that Not4p, a component of the Ccr4-Not complex, may act as an E3 ubiquitin-protein ligase for translation arrest products. The results let us propose that the interaction between basic amino acid residues and the negatively charged exit tunnel of the ribosome leads to translation arrest followed by Not4p-mediated ubiquitination and protein degradation by the proteasome.

Nuclear Import Factor Srp1 and Its Associated Protein Sts1 Couple Ribosome-bound Nascent Polypeptides to Proteasomes for Cotranslational Degradation

“…Early studies suggested that ubiquitylation of nascent polypeptides provides a targeting signal for the proteasome (8, 14 -16). This suggestion is supported by the identification of several ribosome-bound ubiquitin (Ub) 2 ligases, including Not4 and Lin1 (17,18). Interestingly, cotranslational ubiquitylation appears to occur at a low level.…”

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins

“…Translation of the poly(A) tail of an NS mRNA molecule is likely to result in a terminally stalled complex containing a ribosome, an mRNA molecule, and a tRNA-linked protein with a C-terminal polylysine extension (10). In addition to the absence of a stop codon to trigger termination, electrostatic attraction between the translated basic polylysine tract and the acidic ribosome exit tunnel is believed to prolong ribosome occupancy (3,11,12). Indeed, translational pausing and ribosome-associated degradation can be induced by artificially inserting a sequence encoding several consecutive basic amino acids upstream of a wild-type (WT) stop codon (3,11,13).…”

“…His3, protein A, and green fluorescent protein (GFP)) engineered to be translated beyond their stop codons or to possess internal polybasic sequences that mimic a translated poly(A) tail (3,11,13,19,22). Following translational pausing and ribosome dissociation, translationally stalled cytosolic proteins are expected to have their N-terminal portions exposed to the cytosol where the RQC complex would have access (17,23).…”

“…In addition to the absence of a stop codon to trigger termination, electrostatic attraction between the translated basic polylysine tract and the acidic ribosome exit tunnel is believed to prolong ribosome occupancy (3,11,12). Indeed, translational pausing and ribosome-associated degradation can be induced by artificially inserting a sequence encoding several consecutive basic amino acids upstream of a wild-type (WT) stop codon (3,11,13). The heterodimeric complex of Hbs1 (a GTPase with homology to translational release factors) and Dom34 (which adopts a conformation that mimics tRNA structure) stimulates ribosome dissociation into large and small subunits (10, 14 -16), potentially with assistance from the ATPase Rli1 (1).…”

PATACSDB—the database of polyA translational attenuators in coding sequences

“…Over the years, we have got to know that certain sequence features can trigger ribosome stalling. These are damaged bases (Cruz-Vera et al, 2004), stable stem-loop structures (Doma & Parker, 2006), rare codons (Letzring, Dean & Grayhack, 2010), mRNAs lacking stop codons (so called non-stop mRNAs) (Dimitrova et al, 2009), runs of codons that encode consecutive basic aminoacids (Kuroha et al, 2010;Brandman et al, 2012), or finally, runs of adenines encoding poly-lysine tracks Arthur et al, 2015).…”