CoPixie, a novel algorithm for single-particle track colocalization, enables efficient quantification of telomerase dynamics at telomeres.

Single-particle imaging and tracking can be combined with colocalization analysis to study the dynamic interactions between macromolecules in living cells. Indeed, single-particle tracking has been extensively used to study protein-DNA interactions and dynamics. Still, unbiased identification and quantification of binding events at specific genomic loci remains challenging. Herein, we describe CoPixie, a new software that identifies colocalization events between a theoretically unlimited number of imaging channels, including single-particle movies. CoPixie is an object-based colocalization algorithm that relies on both pixel and trajectory overlap to determine colocalization between molecules. We employed CoPixie with live-cell single-molecule imaging of telomerase and telomeres, to test the model that cancer-associated POT1 mutations facilitate telomere accessibility. We show that POT1 mutants Y223C, D224N or K90E increase telomere accessibility for telomerase interaction. However, unlike the POT1-D224N mutant, the POT1-Y223C and POT1-K90E mutations also increase the duration of long-lasting telomerase interactions at telomeres. Our data reveal that telomere elongation in cells expressing cancer-associated POT1 mutants arises from the dual impact of these mutations on telomere accessibility and telomerase retention at telomeres. CoPixie can be used to explore a variety of questions involving macromolecular interactions in living cells, including between proteins and nucleic acids, from multicolor single-particle tracks.

TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends.

The function of TERRA in the regulation of telomerase in human cells is still debated. While TERRA interacts with telomerase, how it regulates telomerase function remains unknown. Here, we show that TERRA colocalizes with the telomerase RNA subunit hTR in the nucleoplasm and at telomeres during different phases of the cell cycle. We report that TERRA transcripts relocate away from chromosome ends during telomere lengthening, leading to a reduced number of telomeric TERRA-hTR molecules and consequent increase in "TERRA-free" telomerase molecules at telomeres. Using live-cell imaging and super-resolution microscopy, we show that upon transcription, TERRA relocates from its telomere of origin to long chromosome ends. Furthermore, TERRA depletion by antisense oligonucleotides promoted hTR localization to telomeres, leading to increased residence time and extended half-life of hTR molecules at telomeres. Overall, our findings indicate that telomeric TERRA transcripts inhibit telomere elongation by telomerase acting in trans, impairing telomerase access to telomeres that are different from their chromosome end of origin.

Phosphorylation controls the oligomeric state of She2 and mRNA localization in yeast.

Messenger RNA (mRNA) localization is an important mechanism controlling local protein synthesis. In budding yeast, asymmetric localization of transcripts such as ASH1 mRNA to the bud tip depends on the She2 RNA-binding protein. She2 assembles as a tetramer to bind RNA, but the regulation of this process as part of the mRNA locasome is still unclear. Here, we performed a phosphoproteomic analysis of She2 in vivo and identified new phosphosites, several of which are located at the dimerization or tetramerization interfaces of She2. Remarkably, phosphomimetic mutations at these residues disrupt the capacity of She2 to promote Ash1 asymmetric accumulation. A detailed analysis of one of these residues, T109, shows that a T109D mutation inhibits She2 oligomerization and its interaction with She3 and the importin-α Srp1. She2 proteins harboring the T109D mutation also display reduced expression. More importantly, this phosphomimetic mutation strongly impairs the capacity of She2 to bind RNA and disrupts ASH1 mRNA localization. These results demonstrate that the control of She2 oligomerization by phosphorylation constitutes an important regulatory step in the mRNA localization pathway.

Quantitative Imaging of MS2-Tagged hTR in Cajal Bodies: Photobleaching and Photoactivation.

Advances in imaging technologies, gene editing, and fluorescent molecule development have made real-time imaging of nucleic acids practical. Here, we detail methods for imaging the human telomerase RNA template, hTR via the use of three inserted MS2 stem loops and cognate MS2 coat protein (MCP) tagged with superfolder GFP or photoactivatable GFP. These technologies enable tracking of the dynamics of RNA species through Cajal bodies and offer insight into their residence time in Cajal bodies through photobleaching and photoactivation experiments. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020).

A single-molecule view of telomerase regulation at telomeres.

Telomerase plays a key role in the immortalization of cancer cells by maintaining telomeres length. Using single-molecule imaging of telomerase RNA molecules in cancer cells, we recently reported novel insights into the role of Cajal bodies in telomerase biogenesis and the regulation of telomerase recruitment to telomeres.

Imaging of Telomerase RNA by Single-Molecule Inexpensive FISH Combined with Immunofluorescence.

Fluorescent in situ hybridization (FISH) on the RNA moiety of human telomerase (hTR) with 50-mer probes detects hTR RNA accumulated in Cajal bodies. Using both live-cell imaging and single-molecule inexpensive FISH, our published work revealed that only a fraction of hTR localizes to Cajal bodies, with the majority of hTR molecules distributed throughout the nucleoplasm. This protocol is an application guide to the smiFISH method for the dual detection of hTR RNA and telomeres or Cajal bodies by immunofluorescence. For complete details on the use and execution of this protocol, please refer to Laprade et al. (2020).

Single-Molecule Imaging of Telomerase RNA Reveals a Recruitment-Retention Model for Telomere Elongation.

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine 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, and 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 reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.

TERRA, a Multifaceted Regulator of Telomerase Activity at Telomeres.

In eukaryotes, telomeres are repetitive sequences at the end of chromosomes, which are maintained in a constitutive heterochromatin state. It is now known that telomeres can be actively transcribed, leading to the production of a telomeric repeat-containing noncoding RNA called TERRA. Due to its sequence complementarity to the telomerase template, it was suggested early on that TERRA could be an inhibitor of telomerase. Since then, TERRA has been shown to be involved in heterochromatin formation at telomeres, to invade telomeric dsDNA and form R-loops, and even to promote telomerase recruitment at short telomeres. All these functions depend on the diverse capacities of this lncRNA to bind various cofactors, act as a scaffold, and promote higher-order complexes in cells. In this review, it will be highlighted as to how these properties of TERRA work together to regulate telomerase activity at telomeres.

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells.

Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

Induction and relocalization of telomeric repeat-containing RNAs during diauxic shift in budding yeast.

Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase. In the budding yeast Saccharomyces cerevisiae, TERRA accumulation is repressed by telomeric silencing and the Rat1 exonuclease. On the other hand, telomere shortening promotes expression of TERRA. So far, little is known about the biological processes that induce TERRA expression in yeast. Understanding the dynamics of TERRA expression and localization is essential to define its function in telomere biology. Here, we aim to study the dynamics of TERRA expression during yeast cell growth. Using live-cell imaging, RNA-FISH and quantitative RT-PCR, we show that TERRA expression is induced as yeast cells undergo diauxic shift, a lag phase during which yeast cells switch their metabolism from anaerobic fermentation to oxidative respiration. This induction is transient as TERRA levels decrease during post-diauxic shift. The increased expression of TERRA is not due to the shortening of telomeres or increased stability of this transcript. Surprisingly, this induction is coincident with a cytoplasmic accumulation of TERRA molecules. Our results suggest that TERRA transcripts may play extranuclear functions with important implications in telomere biology and add a novel layer of complexity in the interplay between telomere biology, metabolism and stress response.

Telomerase RNA Imaging in Budding Yeast and Human Cells by Fluorescent In Situ Hybridization.

Telomerase, the enzyme that elongates telomeres in most eukaryotes, is a ribonucleoprotein complex composed of a reverse transcriptase catalytic subunit (TERT in human, Est2 in the budding yeast S. cerevisiae), regulatory factors and a noncoding RNA called hTERC (in human) or TLC1 (in budding yeast). Telomerase trafficking is a major process in the biogenesis and regulation of telomerase action at telomeres. Due to its higher signal-to-noise ratio, imaging of the telomerase RNA moiety is frequently used to determine telomerase intracellular localization. Here we describe how to image telomerase RNA in human and yeast cells using fluorescence in situ hybridization.

RNA fluorescence in situ hybridization for high-content screening.

Single molecule RNA imaging using fluorescent in situ hybridization (FISH) can provide quantitative information on mRNA abundance and localization in a single cell. There is now a growing interest in screening for modifiers of RNA abundance and/or localization. For instance, microsatellite expansion within RNA can lead to toxic gain-of-function via mislocalization of these transcripts into RNA aggregate and sequestration of RNA-binding proteins. Screening for inhibitors of these RNA aggregate can be performed by high-throughput RNA FISH. Here we describe detailed methods to perform single molecule RNA FISH in multiwell plates for high-content screening (HCS) microscopy. We include protocols adapted for HCS with either standard RNA FISH with fluorescent oligonucleotide probes or the recent single molecule inexpensive FISH (smiFISH). Recommendations for success in HCS microscopy with high magnification objectives are discussed.

Cell cycle-dependent spatial segregation of telomerase from sites of DNA damage.

Telomerase can generate a novel telomere at DNA double-strand breaks (DSBs), an event called de novo telomere addition. How this activity is suppressed remains unclear. Combining single-molecule imaging and deep sequencing, we show that the budding yeast telomerase RNA (TLC1 RNA) is spatially segregated to the nucleolus and excluded from sites of DNA repair in a cell cycle-dependent manner. Although TLC1 RNA accumulates in the nucleoplasm in G1/S, Pif1 activity promotes TLC1 RNA localization in the nucleolus in G2/M. In the presence of DSBs, TLC1 RNA remains nucleolar in most G2/M cells but accumulates in the nucleoplasm and colocalizes with DSBs in rad52Δ cells, leading to de novo telomere additions. Nucleoplasmic accumulation of TLC1 RNA depends on Cdc13 localization at DSBs and on the SUMO ligase Siz1, which is required for de novo telomere addition in rad52Δ cells. This study reveals novel roles for Pif1, Rad52, and Siz1-dependent sumoylation in the spatial exclusion of telomerase from sites of DNA repair.

Live-cell imaging of budding yeast telomerase RNA and TERRA.

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs.

Smc5/6 Is a Telomere-Associated Complex that Regulates Sir4 Binding and TPE.

SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). SIR4 recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, nse3-1/ sir4 Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation.

Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro.

AIM: Recent evidence shows that localization of mRNAs and their protein products at cellular protrusions plays a decisive function in the metastasis of cancer cells. The aim of this study was to identify the variety of proteins encoded by protrusion-localized mRNAs and their roles in the metastasis and invasion of liver cancer cells. METHODS: Highly metastatic hepatocellular carcinoma cell line HCCLM3 and non-metastatic hepatocellular carcinoma cell line SMMC-7721 were examined. Cell protrusions (Ps) were separated from cell bodies (CB) using a Boyden chamber assay; total mRNA population in CB and Ps fractions was analyzed using high-throughput direct RNA sequencing. The localization of STAT3 mRNA and protein at Ps was confirmed using RT-qPCR, RNA FISH, and immunofluorescence assays. Cell migration capacity and invasiveness of HCCLM3 cells were evaluated using MTT, wound healing migration and in vitro invasion assays. The interaction between Stat3 and growth factor receptors was explored with co-immunoprecipitation assays. RESULTS: In HCCLM3 cells, 793 mRNAs were identified as being localized in the Ps fraction according to a cut-off value (Ps/CB ratio) >1.6. The Ps-localized mRNAs could be divided into 4 functional groups, and were all closely related to the invasive and metastatic properties. STAT3 mRNA accumulated in the Ps of HCCLM3 cells compared with non-metastatic SMMC-7721 cells. Treatment of HCCLM3 cells with siRNAs against STAT3 mRNA drastically decreased the cell migration and invasion. Moreover, Ps-localized Stat3 was found to interact with pseudopod-enriched platelet-derived growth factor receptor tyrosine kinase (PDGFRTK) in a growth factor-dependent manner. CONCLUSION: This study reveals STAT3 mRNA localization at the Ps of metastatic hepatocellular carcinoma HCCLM3 cells by combining application of genome-wide and gene specific description and functional analysis.

Telomeric repeat-containing RNA TERRA: a noncoding RNA connecting telomere biology to genome integrity.

Telomeres are dynamic nucleoprotein structures that protect the ends of chromosomes from degradation and activation of DNA damage response. For this reason, telomeres are essential to genome integrity. Chromosome ends are enriched in heterochromatic marks and proper organization of telomeric chromatin is important to telomere stability. Despite their heterochromatic state, telomeres are transcribed giving rise to long noncoding RNAs (lncRNA) called TERRA (telomeric repeat-containing RNA). TERRA molecules play critical roles in telomere biology, including regulation of telomerase activity and heterochromatin formation at chromosome ends. Emerging evidence indicate that TERRA transcripts form DNA-RNA hybrids at chromosome ends which can promote homologous recombination among telomeres, delaying cellular senescence and sustaining genome instability. Intriguingly, TERRA RNA-telomeric DNA hybrids are involved in telomere length homeostasis of telomerase-negative cancer cells. Furthermore, TERRA transcripts play a role in the DNA damage response (DDR) triggered by dysfunctional telomeres. We discuss here recent developments on TERRA's role in telomere biology and genome integrity, and its implication in cancer.