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Messenger RNA (mRNA) localization is an important mechanism controlling local protein synthesis. In budding yeast, asymmetric localization of transcripts such as ASH1mRNA 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 ASH1mRNA localization. These results demonstrate that the control of She2 oligomerization by phosphorylation constitutes an important regulatory step in the mRNA localization pathway.

ABSTRACTTelomeres 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.

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.

Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and activation of DNAdamage response. For this reason, functional telomeres are vital to genome stability. For years, telomeres were assumed to be transcriptionally silent, because of their heterochromatic state. It was only recently shown that, in several organisms, telomeres are transcribed, giving rise to a long noncoding RNA(lncRNA) called telomeric repeat‐containing RNA (TERRA). Several lines of evidence now indicate that TERRAmolecules play crucial roles in telomere homeostasis and telomere functions. Recent studies have shown that the expression and regulation of TERRAare dynamically controlled by each chromosome end. TERRAhas been involved in the regulation of telomere length, telomerase activity, and heterochromatin formation at telomeres. The correct regulation of the telomeric transcripts may be essential to genome stability, and altered TERRAlevels associate with tumorigenesis and cellular senescence. Thus, the study of the molecular mechanisms of TERRAbiogenesis and function may advance the understanding of telomere‐related diseases, including cancer and aging.

The stable linearity of eukaryotic chromosomes depends on special characteristics of their ends, the telomeres. Accurate telomere function in turn requires a sustained presence of repeated DNA elements, which are maintained by the enzyme telomerase. The telomerase holoenzyme is composed of both protein and RNA, and its functions rely on proper expression, maturation, trafficking and assembly of these components. Conflicting models for the recruitment of telomerase at telomeres have been proposed; one suggests a local activation of telomerase at short telomeres, while the other proposes that telomerase is recruited only at short telomeres. To discriminate between these models and investigate the cell cycle-dependent regulation of telomerase in living cells, a GFP reporter system to visualize the yeast telomerase RNA has been recently developed. This assay shed new light on the mechanism of recruitment of telomerase to telomeres, and it uncovered a hitherto unrecognized mechanism for restricting telomerase access to telomeres.

Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence have revealed that the full picture is more complex, and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA.

The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleocytoplasmic shuttling protein She2 binds the ASH1mRNA and targets it for localization at the bud tip by recruiting the She3p–Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is essential for its nuclear import via the importin α Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS leads to defective ASH1mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1mRNA and control its translation. We find that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction. Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1mRNA, suggesting that nuclear import of She2p is necessary to recruit both factors to the ASH1transcript. This study reveals that a direct coupling between localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.

The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleocytoplasmic shuttling protein She2 binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p–Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is essential for its nuclear import via the importin α Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS leads to defective ASH1 mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1 and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1 mRNA and control its translation. We find that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction. Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1 mRNA, suggesting that nuclear import of She2p is necessary to recruit both factors to the ASH1 transcript. This study reveals that a direct coupling between localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.

Les micro-ARN représentent une découverte majeure dans le domaine de la régulation de l’expression des gènes. Ils participent à la régulation de l’expression de protéines en inhibant la traduction et/ou en induisant la dégradation de leurs ARNm correspondants. L’étude de leurs fonctions a révélé leur rôle dans la prolifération cellulaire et le développement chez les métazoaires. La recherche des altérations moléculaires en cause dans l’apparition de cancers a permis d’identifier ces molécules en tant que nouveaux acteurs de la transformation néoplasique. Ainsi, des micro-ARN présentant les caractéristiques d’oncogènes (miR-17-92) ou celles de suppresseur de tumeurs (let-7) ont été découverts ces dernières années.

Telomere maintenance in eukaryotes is linked to cellular proliferation capacity. The telomerase holoenzyme, which maintains telomere length, is a central actor in this process and its function is highly conserved among eukaryotes. Telomerase is a ribonucleoproteic complex constituted of an RNA template and proteic subunits with catalytic and regulatory activities. Several factors required for the activity and recruitment of telomerase at telomeres have been identified in the past years. However, it is still poorly understood how this holoenzyme is assembled in cells. Recent studies in mammalian and yeast cells have underscored the importance of telomerase RNA trafficking in telomerase biogenesis and telomere homeostasis. These studies revealed that telomerase biogenesis is a highly dynamic process, which is regulated in space and time, and which ultimately affects how cells maintain genome integrity.

ABSTRACTThe cyc1-512mutation in Saccharomyces cerevisiaecauses a 90% reduction in the level of iso-1-cytochrome cbecause of the lack of a proper 3'-end-forming signal, resulting in low levels of eight aberrantly longcyc1-512mRNAs which differ in length at their 3' termini. cyc1-512can be suppressed by deletion of either of the nonessential genes CBC1and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-? deletion suppressed the cyc1-512defect by diminishing degradation of the longer subset ofcyc1-512mRNAs, suggesting that downstream elements or structures occurred in the extended 3' region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512defects by cbc1-? occurred by two different mechanisms. The levels of the shorter cyc1-512transcripts were enhanced in the cbc1-? mutants by promoting 3'-end formation at otherwise-weak sites, whereas the levels of the longercyc1-512transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore,cbc1-? greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

The cyc1-512mutation in Saccharomyces cerevisiaecauses a 90% reduction in the level of iso-1-cytochrome cbecause of the lack of a proper 3′-end-forming signal, resulting in low levels of eight aberrantly longcyc1-512mRNAs which differ in length at their 3′ termini. cyc1-512can be suppressed by deletion of either of the nonessential genes CBC1and CBC2, which encode the CBP80 and CBP20 subunits of the nuclear cap binding complex, respectively, or by deletion of the nonessential gene UPF1, which encodes a major component of the mRNA surveillance complex. The upf1-Δ deletion suppressed the cyc1-512defect by diminishing degradation of the longer subset of cyc1-512mRNAs, suggesting that downstream elements or structures occurred in the extended 3′ region, similar to the downstream elements exposed by transcripts bearing premature nonsense mutations. On the other hand, suppression of cyc1-512defects by cbc1-Δ occurred by two different mechanisms. The levels of the shorter cyc1-512transcripts were enhanced in the cbc1-Δ mutants by promoting 3′-end formation at otherwise-weak sites, whereas the levels of the longercyc1-512transcripts, as well as of all mRNAs, were slightly enhanced by diminishing degradation. Furthermore,cbc1-Δ greatly suppressed the degradation of mRNAs and other phenotypes of a rat7-1strain which is defective in mRNA export. We suggest that Cbc1p defines a novel degradation pathway that acts on mRNAs partially retained in nuclei.

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.

Fluorescent in situhybridization (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.

A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer.