Non-coding RNA, Epigenetics, and Genomes Fluidity

Fig 1. The destiny of aslncRNAs in yeast. A cytoplasmic aslncRNA can associate with its paired-sense mRNA to form an RNA:RNA duplex (1), that can be subsequently dissociated by RNA helicases. Alternatively, the aslncRNA can be directly bound by ribosomes, translated and targeted to Xrn1 through the NMD (2). The checkpoints dictating the fate of this aslncRNA and the competition between duplex formation and translation remain to be defined.

The pervasive transcription of genomes produces thousands of long noncoding (lnc)RNAs, including antisense (as)lncRNAs synthesized from the DNA strand opposite to protein-coding genes. Despite their regulatory potential, aslncRNAs remain poorly characterized. This is probably due to their low cellular abundance, as they are extensively degraded by nuclear and cytoplasmic RNA surveillance pathways. Our recent works in yeast revealed that cytoplasmic aslncRNAs are mainly targeted by the Nonsense-Mediated Decay (NMD), a translation-dependent RNA decay pathway known to degrade abnormal mRNAs with premature translation termination codon, suggesting that aslncRNAs are translated. Conversely, aslncRNAs can form double-stranded (ds)RNA with their paired-sense mRNAs, and this modulates their sensitivity to the NMD, indicating that the dynamics of pairing is crucial for their decay/expression. However, the extent, the decision point and the regulatory mechanisms determining the fate of the lncRNA either subjected to translation/decay or pairing/stabilization are unknown (Fig 1). To tackle these questions, our project aims at defining:

- the translational control and the output (lncRNA-derived peptides) of the yeast ‘noncoding’ transcriptome;

- the decision point modulating the heterogeneity of aslncRNAs/mRNAs tandem expression and duplex formation at the single-cell level;

- the impact of dsRNA formation on the post-transcriptional metabolism of the interacting transcripts.

Our collaborators :

# Daniel Gautheret (I2BC, Gif sur Yvette, France)

# Olivier Namy (I2BC, Gif sur Yvette, France)

# Damien Hermand (Univ de Namur, Belgian)

# Manolis Papamichos (Univ of Liverpool, UK)

Figure 2: LncRNA in cancer biology

High-throughput total RNA sequencing in humans produced catalogues of lncRNAs  whose number nowadays exceeds those of protein-coding genes reaching more than 100 000 transcripts according to LNCipedia (Volders, 2015). Growing number of lncRNAs were shown to accomplish a remarkable variety of biological functions including gene expression regulation, maintenance of genome integrity and defense hence contributing to normal and pathological development at cellular and organismal levels. Importantly, lncRNA expression is tightly regulated and often perturbed in cancers highlighting not only cancer type specific patterns but also broad intratumor heterogeneity. All these features promote lncRNAs as attractive candidates to study their role in cell plasticity and cell-to-cell communication during cancer initiation, progression and response to treatment.

We aim to identify novel lncRNAs associated with different types of cancers applying RNA-sequencing and original computational analysis of clinical specimens (prostate cancer, melanoma, triple negative breast cancer). In addition to bulk RNA-sequencing of solid tumors we perform broad transcriptome analysis of urine and blood derived extracellular vesicles (EVs) to define RNA species secreted by tumors with a possible role in cell-to-cell communication and remodeling of tumor microenvironment. Newly discovered lncRNAs are further studied for their protein partners and function using various in vitro cell systems and genetic tools (CRISPR, ASO, siRNA tools). Applying single-molecule RNA-FISH and CUT&RUN approaches we aim to decipher lncRNAs’ molecular mechanisms of action with a specific focus on epigenetic and transcriptomic reprogramming of cells in response to alterations in lncRNA expression.

Current projects:

1. Functional characterization of lncRNAs associated with neuroendocrine trans-differentiation and acquisition of androgen-independence in prostate cancer.
2. LncRNAs involved in epithelial-mesenchymal transition.
3. LncRNA fate in cytoplasm and role in multiple myeloma.
4. Discovering functional lncRNAs in neoadjuvant chemoresistant triple negative breast cancer.

Our collaborators :

# Francis Vacherot, Virginie Firlej et Damien Destouches (TRePCa - Résistances Thérapeutiques du Cancer de la Prostate, UPEC – UFR de Santé, Créteil)

# Céline Vallot (Dynamique de la plasticité épigénétique dans le cancer, UMR 3244, Institut Curie, Paris)

# Daniel Gautheret (I2BC, Gif sur Yvette, France)

# Olivier Namy (I2BC, Gif sur Yvette, France)

# Janusz Bujnicki (IMMB, Varsaw, Poland)

# Fernando Moreno Herrero (CNB, Madrid, Spain)

# Oscar Llorca (CIO, Madrid Spain)

# Nolwenn Jouvenet (Pasteur, Paris)

Figure 3: RNA biomarkers in extracellular vesicles

Extracellular vesicles (EVs), such as exosomes, microvesicles, or oncosomes, are secreted membrane-enclosed vesicles produced by all types of cells. EVs carry distinct repertoires of proteins, lipids, metabolites and nucleic acids (DNA and RNA), and their cargo is controlled by specific molecular sorting machineries. EVs coming from tumor cells can circulate in fluids like plasma and urine and can vehicle biomarkers protected from potential degradation by nucleases, proteases or other environmental stressors. Consequently, EVs can be an excellent material to help for a better understanding of cancer biology and to discover novel biomarkers for cancer diagnostics and prognosis and for therapeutic options.

To define novel RNA biomarkers for prostate cancer prognostic, we analyze the full transcriptome of urinary and blood extracellular vesicles from healthy and prostate cancer (PCa) patients and compare it with prostate tumor tissues transcriptome. Finally, we orientate future functional studies of selected PCa-EVs biomarkers using prostate cell lines that produce EVs carrying these biomarkers.

Current projects:

1. Comparison of urinary EVs and tumor cells RNA cargoes from prostate cancer patients.
2. Clinical relevance of urine and blood derived extracellular vesicles (EVs) to define RNA biomarkers for non-invasive prognosis of prostate cancer.
3. Functional study of selected urinary and blood EVs biomarkers from prostate cancer patients.

Our collaborators :

# Clotilde Théry (institut Curie, France)

# Francis Vacherot, Virginie Firlej et Damien Destouches (TRePCa - Résistances Thérapeutiques du Cancer de la Prostate, UPEC – UFR de Santé, Créteil, FRance)

# Daniel Gautheret (I2BC, Gif sur Yvette, France)

# Prof Alexandre de la taille (Mondor hospital, Creteil, France)

# Prof Yves Allory (Head of Pathology, Institut Curie, St-Cloud & Hôpital Foch, Suresnes, France)