Super-telomeres in transformed human fibroblasts

Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis

Visual Search of Neuropil-Enriched RNAs from Brain In Situ Hybridization Data through the Image Analysis Pipeline Hippo-ATESC

International audienceMotivation: RNA molecules specifically enriched in the neuropil of neuronal cells and in particular in dendritic spines are of great interest for neurobiology in virtue of their involvement in synaptic structure and plasticity. The systematic recognition of such molecules is therefore a very important task. High resolution images of RNA in situ hybridization experiments contained in the Allen Brain Atlas (ABA) represent a very rich resource to identify them and have been so far exploited for this task through human-expert analysis. However, software tools that may automatically address the same objective are not very well developed. Results: In this study we describe an automatic method for exploring in situ hybridization data and discover neuropil-enriched RNAs in the mouse hippocampus. We called it Hippo-ATESC (Automatic Texture Extraction from the Hippocampal region using Soft Computing). Bioinformatic validation showed that the Hippo-ATESC is very efficient in the recognition of RNAs which are manually identified by expert curators as neuropil-enriched on the same image series. Moreover, we show that our method can also highlight genes revealed by microdissection-based methods but missed by human visual inspection. We experimentally validated our approach by identifying a non-coding transcript enriched in mouse synaptosomes. The code is freely available on the web at http://ibislab.ce.unipr.it/software/hippo/

Functional characterization of the AFF (AF4/FMR2) family of RNA-binding proteins: insights into the molecular pathology of FRAXE intellectual disability.

International audienceThe AFF (AF4/FMR2) family of genes includes four members: AFF1/AF4, AFF2/FMR2, AFF3/LAF4 and AFF4/AF5q31. AFF2/FMR2 is silenced in FRAXE intellectual disability, while the other three members have been reported to form fusion genes as a consequence of chromosome translocations with the myeloid/lymphoid or mixed lineage leukemia (MLL) gene in acute lymphoblastic leukemias (ALLs). All AFF proteins are localized in the nucleus and their role as transcriptional activators with a positive action on RNA elongation was primarily studied. We have recently shown that AFF2/FMR2 localizes to nuclear speckles, subnuclear structures considered as storage/modification sites of pre-mRNA splicing factors, and modulates alternative splicing via the interaction with the G-quadruplex RNA-forming structure. We show here that similarly to AFF2/FMR2, AFF3/LAF4 and AFF4/AF5q31 localize to nuclear speckles and are able to bind RNA, having a high apparent affinity for the G-quadruplex structure. Interestingly, AFF3/LAF4 and AFF4/AF5q31, like AFF2/FMR2, modulate, in vivo, the splicing efficiency of a mini-gene containing a G-quadruplex structure in one alternatively spliced exon. Furthermore, we observed that the overexpression of AFF2/3/4 interferes with the organization and/or biogenesis of nuclear speckles. These findings fit well with our observation that enlarged nuclear speckles are present in FRAXE fibroblasts. Furthermore, our findings suggest functional redundancy among the AFF family members in the regulation of splicing and transcription. It is possible that other members of the AFF family compensate for the loss of AFF2/FMR2 activity and as such explain the relatively mild to borderline phenotype observed in FRAXE patients

List of the top-scoring 20 protein-coding genes defined by the pipeline.

<p>Gene names marked by a star correspond to genes mapping to the mitochondrial genome. ABA indicates the Allen Brain Atlas experiment number. In the SF and MF columns, the genes which were previously associated to dendritic spine or synaptic function and the genes associated to mitochondrial function are marked, respectively.</p

Schematic representation of the Hippo-ATESC pipeline.

<p>Schematic representation of the Hippo-ATESC pipeline.</p

Boxplot of the correlation values of the feature vectors obtained from 6 slices of 8 randomly selected genes, as compared to the prototype vector.

<p>Note the very stable behavior of genes characterized by a high Pearson coefficient.</p

Relative expression in adult brain and synaptosomes of the top-scoring non-coding RNAs identified by the Hippo-ATESC pipeline, as determined by qRT-PCR.

<p>In the last column is reported the ratio between synaptosomal and total brain levels. A.U. = arbitrary units.</p

New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes

International audienceCytoplasmic FMRP interacting protein 1 (CYFIP1) is a candidate gene for intellectual disability (ID), autism, schizophrenia and epilepsy. It is a member of a family of proteins that is highly conserved during evolution, sharing high homology with its Drosophila homolog, dCYFIP. CYFIP1 interacts with the Fragile X mental retardation protein (FMRP, encoded by the FMR1 gene), whose absence causes Fragile X syndrome, and with the translation initiation factor eIF4E. It is a member of the WAVE regulatory complex (WRC), thus representing a link between translational regulation and the actin cytoskeleton. Here, we present data showing a correlation between mRNA levels of CYFIP1 and other members of the WRC. This suggests a tight regulation of the levels of the WRC members, not only by post-translational mechanisms, as previously hypothesized. Moreover, we studied the impact of loss of function of both CYFIP1 and FMRP on neuronal growth and differentiation in two animal models – fly and mouse. We show that these two proteins antagonize each other's function not only during neuromuscular junction growth in the fly but also during new neuronal differentiation in the olfactory bulb of adult mice. Mechanistically, FMRP and CYFIP1 modulate mTor signaling in an antagonistic manner, likely via independent pathways, supporting the results obtained in mouse as well as in fly at the morphological level. Collectively, our results illustrate a new model to explain the cellular roles of FMRP and CYFIP1 and the molecular significance of their interaction