Biology subjects

Cheng, R. C.

Publications and source records attributed to Cheng, R. C..

3 recordsLinked to original sources

bioRxiv2025

Tight translation regulation of the canonical C9ORF72 through a multi-component system

C9ORF72 is involved in multiple neuronal functions and a major factor in Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disease. Pathogenicity is implemented through an intronic repeat expansion in the genes mRNA leader that produces long, repetitive RNA and dipeptides when translated through a non-canonical mechanism. In contrast, despite the presence of ribosome-occupied, regulatory elements in the mRNA leader, nothing is known about C9ORF72 translation regulation under normal conditions. Surprisingly, when analyzing a series of mutants of the C9ORF72 mRNA leader, we found that translation of C9ORF72 is tightly regulated through a multi-layer system. First, non-canonical, multi-initiation upstream open reading frames (nc-uORFs) in all three frames, a start-stop element in frame 2, and a canonical uORF in frame 1work together to keep baseline C9ORF72 translation very low. Second, a strong secondary structure enhances these repressive elements, mainly the two nc-uORFs in frames 0 and 2. Finally, as initiation at the nc-uORFs reduces initiation at the downstream start-stop, the nc-uORFs effectively dampen the start-stops repressive function, forming a feedforward loop. We hypothesize that this buffered repressor system has likely evolved to ensure reliable, noise-insensitive expression of this critical regulator of neuronal function.

molecular biology↗

bioRxiv2020

Single-molecule FRET monitors CLC transporter conformation and subunit independence

"CLC" transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion access to and from the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Forster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 [A]) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process. SUMMARYCheng, Krishnamoorti et al. use single-molecule Forster energy resonance transfer measurements to monitor the conformation of a CLC transporter and to show that the conformational state is not influenced by the neighboring subunit.

biophysics↗

bioRxiv2019

Structural characterization of an intermediate reveals a unified mechanism for the CLC Cl-/H+ transport cycle

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize many experimental findings, there have remained gaps and inconsistencies in our understanding. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 [A] structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to a state that has improved accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Based on this new structure, together with DEER measurements, MD simulations, and functional studies, we propose a unified model of the CLC transport mechanism that reconciles existing data on all CLC-type proteins.

biophysics↗