Single molecule tracking reveals spatio-temporal dynamics of bacterial DNA repair centres

AbstractThe spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.

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Single-molecule imaging of telomerase RNA reveals a Recruitment – Retention model for telomere elongation

10.1101/2020.01.31.929026 ◽

2020 ◽

Cited By ~ 1

Author(s):

Hadrien Laprade ◽

Emmanuelle Querido ◽

Michael J. Smith ◽

David Guérit ◽

Hannah Crimmins ◽

...

Keyword(s):

Cancer Cells ◽

Single Molecule ◽

Cellular Level ◽

Telomerase Rna ◽

Single Molecule Imaging ◽

Retention Model ◽

Single Molecule Tracking ◽

Time Dynamics ◽

Telomere Elongation ◽

Spatio Temporal

AbstractExtension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatio-temporal coordination of telomerase and telomeres, and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combined CRISPR genome editing and MS2 RNA-tagging to image single-molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, then base-pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life-cycle of telomerase RNA and provides a framework to understand how cancer-associated mutations mechanistically drive defects in telomere homeostasis.

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Single-molecule Tracking Reveals Multi-state Dynamics of a Bacterial DNA Methyltransferase in Vivo

Microscopy and Microanalysis ◽

10.1017/s1431927620018632 ◽

2020 ◽

Vol 26 (S2) ◽

pp. 1590-1591

Author(s):

Ziyuan Chen ◽

Taylor Nye ◽

Lyle Simmons ◽

Julie Biteen

Keyword(s):

Single Molecule ◽

Dna Methyltransferase ◽

Single Molecule Tracking ◽

Bacterial Dna

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Single molecule tracking reveals functions for RarA at replication forks but also independently from replication during DNA repair in Bacillus subtilis

Scientific Reports ◽

10.1038/s41598-018-38289-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

Author(s):

Hector Romero ◽

Thomas C. Rösch ◽

Rogelio Hernández-Tamayo ◽

Daniella Lucena ◽

Silvia Ayora ◽

...

Keyword(s):

Dna Repair ◽

Bacillus Subtilis ◽

Single Molecule ◽

Replication Forks ◽

Single Molecule Tracking

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Nuclear compartmentalization of TERT mRNA and TUG1 lncRNA transcripts is driven by intron retention: implications for RNA-directed therapies

10.1101/2020.07.21.212514 ◽

2020 ◽

Author(s):

Gabrijela Dumbović ◽

Ulrich Braunschweig ◽

Heera K. Langner ◽

Katarzyna Jastrzebska ◽

Michael Smallegan ◽

...

Keyword(s):

Single Molecule ◽

Temporal Dynamics ◽

Intron Retention ◽

Single Cells ◽

Bimodal Distribution ◽

Protein Coding ◽

Rna Molecules ◽

Global Connections ◽

Spatio Temporal ◽

Nuclear Compartmentalization

AbstractNumerous global connections have been made between splicing and other layers of gene regulation, including the spatial partitioning of the transcriptome in the cell. Yet, there has been surprisingly little analysis of the spatio-temporal regulation of individual protein-coding and non-coding RNA molecules in single cells. Here we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH revealed that nuclear TERT transcripts uniformly and robustly retain two specific introns whose splicing occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution had significant effects on cell growth. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of RNAs within the cell; this process reveals new targets for RNA-based therapies.

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Spatio-Temporal Coordination of Transcription Preinitiation Complex Assembly in Live Cells

10.1101/2020.12.30.424853 ◽

2020 ◽

Author(s):

Vu Q. Nguyen ◽

Anand Ranjan ◽

Sheng Liu ◽

Xiaona Tang ◽

Yick Hin Ling ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Chromatin Remodeling ◽

Single Molecule ◽

Transcription Initiation ◽

Live Cells ◽

Gene Promoters ◽

Single Molecule Tracking ◽

Pol Ii ◽

Temporal Model ◽

Spatio Temporal

SUMMARYTranscription 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 Mediator’s 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.

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