Mechanical Forces Regulate the Composition and Fate of Stalled Nascent Chains

Ribosomal quality control failures lead to the accumulation of defective ribosomes and aberrant proteins, causing a range of human diseases and neurodegenerative disorders. These include bone marrow failure syndromes (e.g., Diamond-Blackfan anemia), cancer, neurodegeneration (e.g., Alzheimer’s), and developmental issues. Ribosomal CAT tailing is a Ribosome-associated Quality Control (RQC) mechanism where the protein Rqc2 (NEMF in mammals) adds C-terminal alanine and threonine residues (CAT tails) to stalled polypeptides. The alanine and threonine residues tag the incomplete protein for degradation by the proteasome, helping to prevent the accumulation of toxic protein aggregates. In a recent publication (link), members of the Brandman Lab (Biochemistry) describe how mechanical forces control both the composition and fate of stalled, incompletely translated polypeptides, and how these forces could be manipulated to develop new therapeutics.

Defective mRNA, poly-A sequences, or other signals can prematurely stall translating ribosomes. Trailing ribosomes then collide with these stalled complexes which are subsequently recognized by translational surveillance factors. These factors disassemble the complex, leaving a 60S ribosomal subunit still bound to the peptidyl‑tRNA and incompletely translated polypeptide chain. RQC components Rqc2 and Ltn1 then bind these 60S particles. Rqc2 uses charged Ala‑tRNA and Thr‑tRNA to extend the C terminus of the polypeptide chain in an mRNA‑independent manner, adding a mixed Ala/Thr CAT tail. Because Thr residues interrupt poly‑Ala segments, they prevent stable polyalanine α‑helices from forming inside the 60S exit tunnel, keeping the tail extended, pushing the upstream portion of the nascent chain out of the 60S exit tunnel. The E3 ubiquitin ligase Ltn1 localizes near the ribosomal exit tunnel to ubiquitylate exposed lysine residues on the nascent polypeptide. While in this state, the nascent chain is not under substantial mechanical load, and the RQC machinery is in what the authors term “extrusion mode”.

Once enough lysines are ubiquitylated, the AAA+ ATPase Cdc48 is recruited to the ubiquitylated polypeptide and begins to pull on it. This pulling force is magnified by electrostatic interactions occurring inside the ribosomal exit tunnel, and by pulling caused by cotranslational folding of the recently extruded nascent polypeptide. These forces generate mechanical tension along the polypeptide, transmitted from the external portion of the nascent chain, through the CAT tail, back to the peptidyl‑tRNA anchored in the P site and into the peptidyl‑transferase center.

When this pulling force crosses a threshold, CAT tailing changes. The authors describe how the system enters “release mode,” in which the non‑templated elongation now favors incorporation of alanine alone, excluding threonine. These short Ala tails function as potent C‑terminal degrons that are efficiently recognized by downstream ubiquitin‑proteasome factors. The combination of mechanical tension (pulling) and a short Ala CAT tail promotes cleavage of the peptidyl‑tRNA linkage, and the nascent chain, now bearing ubiquitin and a short Ala tail, is released from the 60S subunit. Once released, this CAT‑tailed protein is targeted to and degraded by the proteasome. Freed of its stalled cargo, the 60S ribosomal subunit can then re‑enter the normal ribosome pool and be reused for new rounds of translation. In this way the RQC system preserves the pool of available ribosomal subunits required for translation, and simultaneously ensures that truncated proteins are efficiently degraded.

The model described by the Brandman lab also explains how failures of the RQC system can lead to disease and neurodegeneration. Their data show that if robust pulling never develops, for example if Ltn1 is absent, Cdc48 is not recruited, folding is impaired, or long polyalanine segments in the tunnel decouple the pulling domain from the peptidyl‑transferase center, CAT tailing remains trapped in extrusion mode. Rqc2 continues to add long, Thr‑rich CAT tails that keep pushing the nascent polypeptide chain out of the 60S exit tunnel with an extended Ala/Thr CAT tail. These extended Ala/Thr tails have a strong tendency to aggregate, and the nascent chains remain tethered to 60S subunits instead of being released. The end result is a buildup of clogged large subunits and Thr‑rich protein aggregates, which together impose proteotoxic stress on the cell. Overall, the study provides critical insight into the molecular basis of RQC‑linked diseases and may guide strategies to mitigate the impact of ribosomal stalling in neurodegeneration, cancer, and other diseases.