Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

Function and Regulation of Telomeric Transcripts – FURETT

Telomeres control by transcripts issued from these chromosomal domains

Telomeres protect chromosomes ends and include repeats of a six nucleotides sequence. The research program seeks to describe how transcripts generated from the telomeric regions and which contain these repeats act on the telomeres chromatin structure and also how they can modify transcription of neighbouring genes.

Processing and effects on chromatin of telomeric transcripts

Telomere length is tightly regulated and decreases during aging. Changes to this regulation are associated with genetic diseases, malignancy and cellular senescence.<br /> It is therefore important to identify factors involved in controlling telomere length and function.<br /> From this perspective it was characterized that these chromosomal domains were transcribed into RNAs of different sizes capable of associating with telomeres. Divergent results were reported on the action of these RNAs on the enzyme telomerase, which increases telomere length.<br /> The research program focuses on understanding how these RNAs are involved in normal and pathological function of telomeres.<br /> The main objectives of the work are to better understand how these telomeric RNAs are modified after transcription, how their stability is controlled, and also how they associate with telomeres. We also seek to establish how they modify telomeric chromatin structure and how they regulate the expression of neighbouring genes. A final part of the program involves the transmission of epigenetic information by small RNAs comprising telomeric repeats.<br /> The fundamental knowledge acquired through the realization of this research should be important to better understand the pathologies of aging and cancer.<br />

Part of the research is analyzing the transcripts from the telomeres. This is done by reverse transcription of these RNAs, and then by quantitative amplification of the resulting DNA. Another approach to this is the direct assessment of the amount of these transcripts by hybridization to a radiolabeled probe or a fluorescent group.
To test the influence of certain proteins on these transcripts, we use the technique of RNA interference which eliminates specific proteins in the cell by degradation of their corresponding messenger RNA following the introduction of small RNAs complementary sequence.
Telomere analyses are performed by various microscopy techniques using fluorescent probes to detect proteins and specific DNA or RNA sequences (especially those comprising the telomeric motif).
For the analysis of the influence of the telomeric transcripts on neighbouring gene expression, a genetic system has been developed for introducing a gene encoding a fluorescent test protein in different configurations upstream of a series of telomeric repeats. This system which allows a simple analysis by measuring the fluorescence of cells will allow us to determine the influence of telomeric repeats on transcription.
Analysis of small RNAs containing telomeric motifs which are present in cells of the germ line is performed by various techniques of deep sequencing. Subcellular localization of these transcripts in mouse oocyte is determined by microinjection of these RNAs labeled with a fluorescent group.

Observations performed on these transcripts issued from telomeres and including repeated telomeric motifs have shown that their intracellular amount is maintained at a low level by a protein that is involved in the control of translation initiation and also in the degradation of RNAs including abnormalities such as a premature stop codon. By examining the presence of DNA damage at telomeres, it has also been observed that this protein exerts a protective role. It is interesting to consider this observation with respect to the genomic instability and oncogenic effects resulting from alteration of the expression of this protein, particularly in the case of breast cancer.
In another part of the work, making use of an original genetic system has allowed us to test the effects of telomeric transcripts on gene expression. This approach has shown that these transcripts exert a negative effect on this process.
A specific property of these telomeric transcripts is binding the telomeres. Interestingly, we have been able to show that small RNAs including telomeric motifs exist in sperm cells and were able to bind specific chromosomal territories in the oocyte. This association is stable and is maintained after several divisions of the initial cell. These small RNA might represent of new way of epigenetic information transmission.

Telomere length is tightly regulated and diminishes regularly at each cell division as a consequence of chromosomes replication. Alteration of this control can lead to premature aging. Telomere alteration can also cause genomic defects and chromosomal instability which can drive emergence of cancer cells. Hence to understand how the telomeric transcripts intervene in the normal protective effect of telomeres appears very important to studies concerning several pathologies. The techniques developed in the frame of this research program to study the telomeric transcripts might lead to new methods of prognostic or diagnostic analyses in the fields of cancer and aging. The work conducted on small RNAs containing telomeric repeats might also bring new interesting concepts regarding embryo development.

- INT6 interacts with MIF4GD/SLIP1 and is necessary for efficient histone mRNA translation.
Neusiedler J, Mocquet V, Limousin T, Ohlmann T, Morris C, Jalinot P.
RNA. (2012) 6:1163-77.
- INT6/EIF3E interacts with ATM and is required for proper execution of the DNA damage response in human cells.
Morris C, Tomimatsu N, Richard DJ, Cluet D, Burma S, Khanna KK, Jalinot P.
Cancer Res. (2012) 8:2006-16.
n.b. : These publications are not directly related to studies concerning telomeric transcripts, but report on data obtained by one of the participating team (PJ) which are important to consider with respect to the research program.

Telomeres are essential nucleoprotein structures that protect linear ends of eukaryotic chromosomes from being recognized and processed as double-strand breaks. They are at the heart of important aspects of cell life as proliferation and senescence. These regions were previously thought to be transcriptionnally silent, but it is now established that they generate heterogeneous transcripts, ranging in length from 100 bases up to at least 9 kilobases, which contain essentially UUAGGG repeats. Synthesis of these non-coding RNA named telomeric repeat-containing RNA (TERRA) appears to be initiated in the subtelomeric region and to proceed into the telomeric track. Interestingly these TERRA colocalize with telomeric components in interphase nuclei and are found at the chromosome tips of metaphase chromosomes. This localization of TERRA at telomeres has been shown to be regulated by suppressors with morphogenic defects in genitalia (SMG) and Up Frameshift (UPF) proteins, thereby establishing an unanticipated link between components of the nonsense-mediated mRNA decay (NMD) machinery and ncRNAs function. Alteration of TERRA association with telomeres resulting from depletion of UPF1 or EST1A correlates with telomeric defects as telomere free chromosome ends or sister telomeres. It has also been shown that this telomeric transcripts are downregulated in cancer cells and during the embryonic development. The available data suggest that TERRA might exert an important regulatory role on telomeres by acting on enzymatic activities, particularly telomerase.
These data raise numerous questions on TERRA processing and association with telomeres, as well as on their effects on these important chromosome domains. The goals of our project will be:
1) to better characterize TERRA transcripts processing by cellular factors of the NMD pathway.
2) to examine a possible role of histone H1 variants in the TERRA tethering to telomeres.
3) to analyze how TERRA act on the telomeres chromatin structure.
4) to study their role in the early mouse development.
Concerning TERRA processing, based on several preliminary results we will focus our attention on the INT6/eIF3e protein. We have indeed previously established that this non-core subunit of the EIF3 translation initiation factor selectively acts in the NMD pathway and interacts with UPF1 and UPF2. An effect of this protein which has been shown to intervene in the control of genome stability in fission yeast and human cells on TERRA processing and tethering to the telomeres will therefore be analyzed in details. Concerning association of TERRA with telomeric chromatin, we plan to develop observations showing that depletion of specific histone H1 variants affects telomeres length, as well as expression of specific ncRNAs. Hence we will test whether specific histone H1 variants regulate TERRA expression and also examine whether they can intervene for bridging the TERRA nucleoprotein and the telomeric chromatin. In continuation of this aspect we will analyze formation of heterochromatin at telomeres. They are indeed characterized by heterochromatin marks and exert a negative effect on expression of genes located in the subtelomeric regions. By analogy with what is known for other ncRNAs as Xist, we will seek for a possible effect of TERRA on chromatin organization at telomeres. To this end it will be analyzed whether forced transcription of telomeres affect the telomere position effect (TPE) as well as various heterochromatic marks. Finally to get a better idea of the functional role of TERRA we will analyze their role on telomere length regulation at early stages of mouse development. Injection of RNA mimicking TERRA in fertilized mouse eggs will allow us to determine whether they associate with telomeres and how this event modifies telomeres lengthening. These approaches will help to better understand the function of TERRA in the whole organism, particularly at early stages of development.

Project coordination


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 500,000 euros
Beginning and duration of the scientific project: - 36 Months

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