Biology subjects

Ling, Y. H.

Publications and source records attributed to Ling, Y. H..

4 recordsLinked to original sources

bioRxiv2024

Live-cell single-molecule dynamics of eukaryotic RNA polymerase machineries

Eukaryotic gene expression in the nucleus is orchestrated by three RNA polymerases (RNAP-I, -II, and -III) and associated factors1,2. Despite extensive biochemical, genomic, structural, and imaging studies, the real-time dynamics of these transcription complexes remain obscure. Here, we employ single-molecule tracking in living yeast to assess the physiological kinetics of over 50 representative proteins encompassing all three RNAP machineries. Components of RNAPI and RNAPIII pre-initiation complexes (PICs) engage in long-lived interactions on chromatin, reflecting their roles for constitutive rRNA and tRNA synthesis, in contrast to the transient RNAPII PIC. We further report the dynamics of key components across the RNAPII transcription cycle2-5--factors for upstream regulation, elongation, histone modification, RNAPII C-terminal domain (CTD) modification, RNA processing, and termination--revealing unprecedented insights into the temporal landscape of RNAPII transcription. Strikingly, many elongation factors, previously thought to travel processively with RNAPII, display transient residence times, suggesting highly dynamic interactions rather than constant association. Systematic screening of RNAPII-associated factors shows that truncation of RNAPII-CTD substantially reduces U1 snRNP residence time and decreases intron retention in ribosomal protein genes, providing insights into how CTD length influences co-transcriptional splicing. Our findings establish a framework for dynamic chromatin interactions of RNA polymerase machineries in living cells.

biophysics↗

bioRxiv2023

Disordered C-terminal domain drives spatiotemporal confinement of RNAPII to enhance search for chromatin targets

Efficient gene expression requires RNA Polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in 3D nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. Here, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is required for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation, and reduces transcription bursting. This study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression.

cell biology↗

bioRxiv2023

Transcriptional Regulation of the P-bodies Associated Psoriasis Candidate Gene CCHCR1

The coiled-coil alpha-helical rod protein 1 (CCHCR1) was first identified as a candidate gene in psoriasis and has lately been associated with COVID-19 susceptibility. Located within P-bodies and centrosomes, its exact cellular role and transcriptional control remain largely unknown. Here, we showed that CCHCR1 shares a bidirectional promoter with its neighboring gene, TCF19. This bidirectional promoter is activated by the G1/S-regulatory transcription factor E2F1, and both genes are co-induced during the G1/S transition of the cell cycle. A luciferase reporter assay suggests that the short intergenic sequence, only 287 bp in length, is sufficient for the G1/S induction of both genes, but the expression of CCHCR1 is further enhanced by the presence of exon 1 from both TCF19 and CCHCR1. This research uncovers the transcriptional regulation of the CCHCR1 gene, offering new perspectives on its function. These findings contribute to the broader understanding of diseases associated with CCHCR1 and may serve as a foundational step for future research in these vital medical fields.

cell biology↗

bioRxiv2020

Spatio-Temporal Coordination Of Transcription Preinitiation Complex Assembly In Live Cells

Transcription initiation by RNA polymerase II (Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus where thousands of promoters are broadly distributed in chromatin, it is unclear how ten individual components converge on any target to establish the PIC. Here, we use live-cell, single-molecule tracking in S. cerevisiae to document subdiffusive, constrained exploration of the nucleoplasm by PIC components and Mediators key functions in guiding this process. On chromatin, TBP, Mediator, and Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently at an average promoter where initiation-coupled disassembly may occur within a few seconds. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Collectively, our study provides a global spatio-temporal model for transcription initiation in live cells.

molecular biology↗