ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Telomerase , Telomere , Telomerase/metabolism , Telomerase/genetics , Humans , Telomere/metabolism , Telomere/genetics , Telomere Homeostasis , HeLa Cells , RNA/metabolism , RNA/genetics , Transcription, Genetic , Telomere-Binding Proteins/metabolism , Telomere-Binding Proteins/genetics , Cell Cycle/genetics , Chromosomes, Human/metabolism , Chromosomes, Human/genetics , DNA-Binding Proteins , Transcription FactorsABSTRACT
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.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Phosphorylation , RNA/metabolismABSTRACT
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).
Subject(s)
Coiled Bodies/metabolism , RNA/analysis , Single Molecule Imaging/methods , Telomerase/analysis , ADAM Proteins/genetics , ADAM Proteins/metabolism , HeLa Cells , Humans , In Situ Hybridization, Fluorescence/methods , Inverted Repeat Sequences/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Photobleaching , RNA/genetics , Telomerase/genetics , Telomere/metabolismABSTRACT
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).
Subject(s)
In Situ Hybridization, Fluorescence/methods , RNA/chemistry , Single Molecule Imaging/methods , Telomerase/chemistry , Coiled Bodies/metabolism , Fluorescent Antibody Technique/methods , HeLa Cells , Humans , RNA/genetics , Telomerase/genetics , Telomere/metabolismABSTRACT
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.
Subject(s)
Coiled Bodies/metabolism , DNA, Single-Stranded/metabolism , RNA/metabolism , Single Molecule Imaging/methods , Telomerase/metabolism , Telomere Homeostasis , Telomere/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , DNA, Single-Stranded/genetics , Gene Editing , HeLa Cells , Humans , Mutation , RNA/genetics , Shelterin Complex , Telomerase/genetics , Telomere/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolismABSTRACT
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.
Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , RNA, Long Noncoding/metabolism , RNA, Neoplasm/metabolism , Telomere/metabolism , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , Humans , Microscopy, Fluorescence , Neoplasms/genetics , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , RNA, Long Noncoding/genetics , RNA, Neoplasm/genetics , Telomere/pathologyABSTRACT
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.
Subject(s)
In Situ Hybridization, Fluorescence/methods , Myoblasts/physiology , RNA, Messenger/genetics , RNA/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , RNA/isolation & purification , RNA, Messenger/isolation & purificationABSTRACT
The telomerase, which is composed of both protein and RNA, maintains genome stability by replenishing telomeric repeats at the ends of chromosomes. Here, we use live-cell imaging to follow yeast telomerase RNA dynamics and recruitment to telomeres in single cells. Tracking of single telomerase particles revealed a diffusive behavior and transient association with telomeres in G1 and G2 phases of the cell cycle. Interestingly, concurrent with telomere elongation in late S phase, a subset of telomerase enzyme clusters and stably associates with few telomeres. Our data show that this clustering represents elongating telomerase and it depends on regulators of telomerase at telomeres (MRX, Tel1, Rif1/2, and Cdc13). Furthermore, the assay revealed premature telomere elongation in G1 in a rif1/2 strains, suggesting that Rif1/2 act as cell-cycle dependent negative regulators of telomerase. We propose that telomere elongation is organized around a local and transient accumulation of several telomerases on a few telomeres.
Subject(s)
RNA/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Telomerase/metabolism , Telomere/metabolism , Cell Cycle , Cell Survival , Microscopy, Confocal , RNA/analysis , Saccharomyces cerevisiae/metabolism , Telomerase/analysis , Telomere/chemistry , ThermodynamicsABSTRACT
Transcripts containing expanded CNG repeats, which are found in several neuromuscular diseases, are not exported from the nucleus and aggregate as ribonuclear inclusions by an unknown mechanism. Using the MS2-GFP system, which tethers fluorescent proteins to a specific mRNA, we followed the dynamics of single CUG-repeat transcripts and RNA aggregation in living cells. Single transcripts with 145 CUG repeats from the dystrophia myotonica-protein kinase (DMPK) gene had reduced diffusion kinetics compared with transcripts containing only five CUG repeats. Fluorescence recovery after photobleaching (FRAP) experiments showed that CUG-repeat RNAs display a stochastic aggregation behaviour, because individual RNA foci formed at different rates and displayed different recoveries. Spontaneous clustering of CUG-repeat RNAs was also observed, confirming the stochastic aggregation revealed by FRAP. The splicing factor Mbnl1 colocalized with individual CUG-repeat transcripts and its aggregation with RNA foci displayed the same stochastic behaviour as CUG-repeat mRNAs. Moreover, depletion of Mbnl1 by RNAi resulted in decreased aggregation of CUG-repeat transcripts after FRAP, supporting a direct role for Mbnl1 in CUG-rich RNA foci formation. Our data reveal that nuclear CUG-repeat RNA aggregates are labile, constantly forming and disaggregating structures, and that the Mbnl1 splicing factor is directly involved in the aggregation process.
Subject(s)
RNA, Messenger/genetics , RNA, Messenger/metabolism , Trinucleotide Repeats , Animals , Fluorescence Recovery After Photobleaching/methods , Gene Expression Profiling , Mice , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , Stochastic Processes , Transcription, Genetic , Trinucleotide Repeat ExpansionABSTRACT
This chapter presents the MS2-GFP system, a method to study the trafficking of RNA molecules in living cells. This system is based on two components: a fusion of the MS2 coat protein to a fluorescent protein and a reporter mRNA containing multimers of the RNA stem-loop recognized by the MS2 coat protein. The MS2-GFP protein bound to the RNA stem-loops acts as a beacon that allows the detection of this mRNA within a cell by epifluorescence or confocal microscopy. This chapter focuses on the use of this system in mammalian fibroblast cells and in yeast cells, and discusses several technical considerations of the MS2-GFP system. Detailed protocols for validating the MS2-GFP signal in fixed cells by fluorescent in situ hybridization of the target RNA using fluorophore-labeled oligonucleotide probes are also provided.
Subject(s)
Capsid Proteins , Fibroblasts/metabolism , Green Fluorescent Proteins , RNA Transport , RNA, Messenger/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Base Sequence , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line , Fibroblasts/cytology , Fibroblasts/ultrastructure , Green Fluorescent Proteins/metabolism , In Situ Hybridization, Fluorescence , Levivirus/genetics , Levivirus/metabolism , Molecular Sequence Data , Plasmids , RNA, Messenger/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/ultrastructureABSTRACT
The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1-3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17-92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17-92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3'-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17-92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17-92 cluster. Our data also point toward an anti-apoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17-92 cluster. Altogether, these results suggest that the autoregulation between E2F1-3 and miR-20a is important for preventing an abnormal accumulation of E2F1-3 and may play a role in the regulation of cellular proliferation and apoptosis.
Subject(s)
E2F Transcription Factors/metabolism , MicroRNAs/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Doxorubicin/pharmacology , E2F Transcription Factors/genetics , Feedback, Physiological , Humans , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Models, Chemical , Oligonucleotides, Antisense , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcriptional ActivationABSTRACT
The p53 tumor suppressor promotes cell cycle arrest or apoptosis in response to stress. Previous work suggests that the promyelocytic leukemia gene (PML) can act upstream of p53 to enhance transcription of p53 targets by recruiting p53 to nuclear bodies (NBs). We show that PML is itself a p53 target gene that also acts downstream of p53 to potentiate its antiproliferative effects. Hence, p53 is required for PML induction in response to oncogenes and DNA damaging chemotherapeutics. Furthermore, the PML gene contains p53 binding sites that confer p53 responsiveness to a heterologous reporter and can bind p53 in vitro and in vivo. Finally, cells lacking PML show a reduced propensity to undergo senescence or apoptosis in response to p53 activation, despite the induction of several p53 target genes. These results identify an additional element of PML regulation and establish PML as a mediator of p53 tumor suppressor functions.
Subject(s)
Gene Expression Regulation, Neoplastic , Leukemia, Promyelocytic, Acute/genetics , Neoplasm Proteins/genetics , Nuclear Proteins , Transcription Factors/genetics , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis/genetics , Base Sequence , Binding Sites , Cell Line , Cell Survival/genetics , Cellular Senescence/genetics , Fibroblasts/cytology , Genes, Reporter , Genes, Tumor Suppressor , Genes, p53 , Genes, ras , Humans , Mice , Mice, Knockout , Promoter Regions, Genetic , Promyelocytic Leukemia Protein , Retroviridae/genetics , Signal Transduction , Tumor Suppressor ProteinsABSTRACT
Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.