Chien-Hung Yu

Biomolecular condensates exhibit spatially heterogeneous microenvironments shaped by intertwined physicochemical factors, yet practical small‐molecule tools for mapping these states in living cells remain limited. Here, we report PyLUMI, a bright dipyrene ratiometric probe, optimized by linker engineering to improve fluorescence output under aqueous conditions while preserving excimer‐to‐monomer (E/M) readout capability. Solution‐phase characterization identifies a scaffold with enhanced fluorescence efficiency and an E/M response to coupled polarity–viscosity microenvironmental changes. In recombinant protein condensates, PyLUMI provides substantially increased signal and enables pixel‐resolved visualization of intra‐condensate heterogeneity. In living cells, the probe enables low‐exposure ratiometric imaging of centrosome‐associated condensate compartments and reveals microenvironmental signatures linked to cell‐cycle progression and pathology‐associated centrosome amplification beyond morphological size differences. These results establish PyLUMI as a functional chemical reporter for spatially resolved profiling of integrated physicochemical fingerprints in condensate‐associated compartments.

The conserved ‐1 programmed ribosomal frameshifting (‐1PRF), essential for eubacteria and RNA viruses, is thought to be exclusively regulated during translation elongation. One of the two required ‐1PRF cis ‐acting elements is a frameshifting stimulatory element (FSE), which pauses the elongating ribosome to stimulate ‐1PRF and is consecutively deformed. Consequently, the remaining hypothesis involves the coordination between translation initiation and FSE reformation, which facilitates the ‐1PRF events in trailing ribosomes. Here, we create a system that allows a tunable translation initiation rate based on the MS2 bacteriophage coat protein translation initiation hairpin to assess this conjecture. We discovered a conserved negative correlation between translation initiation rate and ‐1PRF efficiency in eubacteria and mammalian cells. Mechanistic exploration using Escherichia coli ( E. coli ) shows that a higher initiation rate reduces the frequencies of FSE reformation, resulting in lower ‐1PRF efficiency. The role of translation initiation‐mediated ‐1PRF was examined in an E. coli cellular copA/copA(Z) ‐1PRF event. The results showed that a higher translation initiation rate leads to lower ‐1PRF efficiency, sensitizing E. coli to copper stress. Together, these results suggest that translation initiation is an additional mechanism to regulate ‐1PRF to coordinate optimal protein synthesis for cell fitness, further providing the basis to investigate the interplay between translation initiation and elongation.

The RNA exosome complex (EC) is a multi-protein complex responsible for RNA surveillance. Guided by specific adaptor factors, the EC recognizes RNA species as substrates for processing or degradation. Although its basic structure and components are documented, the regulatory mechanisms that enable this fundamental machinery to respond to biological signals remain unclear. Here, we demonstrate that hypoxia-inducible factor 1-alpha (HIF-1α) brings DGCR8 to associate with the EC in an RNA-independent manner and shifts DGCR8’s RNA-binding preference toward small nucleolar RNAs (snoRNAs). This circuit triggers the snoRNA degradation without affecting their transcription or processing, ultimately impairing ribosomal RNA (rRNA) modifications such as pseudouridylation and 2′-O-methylation, compromising rRNA processing, and reducing global translation efficiency. The HIF-1α-mediated reconfiguration of DGCR8 with EC involves the release of DGCR8 and RRP6 from the nucleoli to the nucleoplasm and is conserved across multiple species, including worms and flies. Under conditions where HIF-1α is induced, we found that the MC-to-EC switch dynamically responds to hypoxic conditions and growth factor signals. In conclusion, the discovery of MC-to-EC switch reveals a multifaceted function of HIF-1α in noncoding RNA regulation, also emphasizing its non-transcriptional impact on the DGCR8-EC-mediated snoRNA degradation pathway and its consequential effects on rRNA modifications and translation, providing new insights into RNA homeostasis regulation.

Spinocerebellar ataxia type 36 (SCA36) is a neurodegenerative disease caused by expanded (GGCCTG)n hexanucleotide repeat sequence in the NOP56 gene. While the expanded repeats could transcribe and form toxic RNA foci within neurons, recent evidence indicates that translation of these repeats produces dipeptide repeats (DPR) that contribute to neurotoxicity. The relative impact of hexanucleotide RNA repeats (HRR) and DPR on the neurodegeneration of SCA36 remains unclear. Here, we established a Drosophila SCA36 model to dissect the neurotoxic effects of HRR and DPR. The fly model recapitulates the cellular defects observed in SCA36 patient fibroblasts, validating its relevance for mechanistic study of SCA36. Further engineering the transgenes to express individual DPRs reveal Proline-Glycine-DPR (PG-DPR) as the most potent neurotoxin causing progressive motor and sensory dysfunction. Expressing a series of the SCA36 transgenes with varying HRR lengths demonstrates an age- and length-dependent adult-onset neurodegeneration. Interestingly, sequence modification of the transgenes to exclusively express HRR or DPR alone causes a milder phenotype, indicating both HRR and DPR contribute partially to the pathogenicity of SCA36. Therefore, this model provides a valuable platform for screening drug targeting either HRR- or DPR-mediated toxicity of SCA36. Suppression of the RNA elongation factor SUPT4H1 ortholog reduces RNA foci in cell culture. However, expression level of SUPT4H1 was not changed in SCA36 patient cells. Interestingly, knockdown of the Drosophila SUPT4H1 ortholog or 6-azauridine treatment to suppress RNA transcription aggravates the neurodegenerative phenotypes in both the fly models and patient-derived fibroblasts, highlighting the complex interplay of pathomechanisms in SCA36. These results underscore the need for carefully evaluating the potential side effects when designing therapeutic interventions for SCA36.

Spinocerebellar ataxia type 36 (SCA36) is a neurodegenerative disease caused by expanded (GGCCTG)n hexanucleotide repeat sequence in the NOP56 gene. While the expanded repeats could transcribe and form toxic RNA foci within neurons, recent evidence indicates that translation of these repeats produces dipeptide repeats (DPR) that contribute to neurotoxicity. The relative impact of hexanucleotide RNA repats (HRR) and DPR on the neurodegeneration of SCA36 remains unclear. Here, we established a Drosophila SCA36 model to dissect the neurotoxic effects of HRR and DPR. The fly model recapitulates the cellular defects observed in SCA36 patient fibroblasts, validating its relevance for mechanistic study of SCA36. Expressing a series of the SCA36 transgenes with varying HRR lengths demonstrates an age- and length-dependent adult-onset neurodegeneration. Further engineering the transgenes to express individual DPRs reveal Proline-Glycine-DPR as the most potent neurotoxin causing progressive motor and sensory dysfunction. Interestingly, sequence modification of the transgenes to exclusively express HRR or DPR alone causes a milder phenotype, indicating both HRR and DPR contribute partially to the pathogenicity of SCA36. Therefore, this model provides a valuable platform for drug screening targeting either HRR or DPR-mediated toxicity of SCA36. Remarkably, Spt4 knockdown or 6-azauridine treatment to suppress RNA transcription aggravates the neurodegenerative phenotypes in both the fly models and patient-derived fibroblasts, highlighting the complex interplay of pathogenic mechanisms in SCA36. These results underscore the need for carefully evaluating the potential side effects when designing therapeutic interventions for SCA36. Summary statement: Spinocerebellar ataxia type 36 (SCA36) is a neurodegenerative disease caused by expanded hexanucleotide repeats. This study establishes a Drosophila model to investigate the neurotoxic mechanisms of both RNA toxicity and dipeptide repeat toxicity in SCA36. The findings highlight the complex interplay between these two factors and provide a platform for identifying potential therapeutic targets.

Packaging signals (PSs) of coronaviruses (CoVs) are specific RNA elements recognized by nucleocapsid (N) proteins that direct the selective packaging of genomic RNAs (gRNAs). These signals have been identified in the coding regions of the nonstructural protein 15 (Nsp 15) in CoVs classified under Embecovirus, a subgenus of betacoronaviruses (beta-CoVs). The PSs in other alpha- and beta-CoVs have been proposed to reside in the 5'-proximal regions of gRNAs, supported by comprehensive phylogenetic evidence. However, experimental data remain limited. In this study, we investigated the interactions between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) 5'-proximal gRNA transcripts and N proteins using electrophoretic mobility shift assays (EMSAs). Our findings revealed that the in vitro synthesized 5'-proximal gRNA transcripts of CoVs can shift from a major conformation to alternative conformations. We also observed that the conformer comprising multiple stem-loops (SLs) is preferentially bound by N proteins. Deletions of the 5'-proximal structural elements of CoV gRNA transcripts, SL1 and SL5a/b/c in particular, were found to promote the formation of alternative conformations. Furthermore, we identified RNA-binding peptides from a pool derived from SARS-CoV N protein. These RNA-interacting peptides were shown to preferentially bind to wild-type SL5a RNA. In addition, our observations of N protein condensate formation in vitro demonstrated that liquid-liquid phase separation (LLPS) of N proteins with CoV-5'-UTR transcripts was influenced by the presence of SL5a/b/c. In conclusion, these results collectively reveal previously uncharacterized binding features between the 5'-proximal transcripts of CoV gRNAs and N proteins.