Caenorhabditis elegans screen reveals role of PAR-5 in RAB-11-recycling endosome positioning and apicobasal cell polarity.

Apically enriched Rab11-positive recycling endosomes (Rab11-REs) are important for establishing and maintaining epithelial polarity. Yet, little is known about the molecules controlling trafficking of Rab11-REs in an epithelium in vivo. Here, we report a genome-wide, image-based RNA interference screen for regulators of Rab11-RE positioning and transport of an apical membrane protein (PEPT-1) in C. elegans intestine. Among the 356 screen hits was the 14-3-3 and partitioning defective protein PAR-5, which we found to be specifically required for Rab11-RE positioning and apicobasal polarity maintenance. Depletion of PAR-5 induced abnormal clustering of Rab11-REs to ectopic sites at the basolateral cortex containing F-actin and other apical domain components. This phenotype required key regulators of F-actin dynamics and polarity, such as Rho GTPases (RHO-1 and the Rac1 orthologue CED-10) and apical PAR proteins. Our data suggest that PAR-5 acts as a regulatory hub for a polarity-maintaining network required for apicobasal asymmetry of F-actin and proper Rab11-RE positioning.

Telomerase efficiently elongates highly transcribing telomeres in human cancer cells.

RNA polymerase II transcribes the physical ends of linear eukaryotic chromosomes into a variety of long non-coding RNA molecules including telomeric repeat-containing RNA (TERRA). Since TERRA discovery, advances have been made in the characterization of TERRA biogenesis and regulation; on the contrary its associated functions remain elusive. Most of the biological roles so far proposed for TERRA are indeed based on in vitro experiments carried out using short TERRA-like RNA oligonucleotides. In particular, it has been suggested that TERRA inhibits telomerase activity. We have exploited two alternative cellular systems to test whether TERRA and/or telomere transcription influence telomerase-mediated telomere elongation in human cancer cells. In cells lacking the two DNA methyltransferases DNMT1 and DNMT3b, TERRA transcription and steady-state levels are greatly increased while telomerase is able to elongate telomeres normally. Similarly, telomerase can efficiently elongate transgenic inducible telomeres whose transcription has been experimentally augmented. Our data challenge the current hypothesis that TERRA functions as a general inhibitor of telomerase and suggest that telomere length homeostasis is maintained independently of TERRA and telomere transcription.

Promoting transcription of chromosome ends.

We recently identified CpG island promoters driving transcription of human telomeric repeat-containing RNA (TERRA). This discovery has shaped a new concept in telomere biology, where TERRA promoters and downstream telomeric sequences constitute autonomous genic units.

CpG-island promoters drive transcription of human telomeres.

The longstanding dogma that telomeres, the heterochromatic extremities of linear eukaryotic chromosomes, are transcriptionally silent was overturned by the discovery that DNA-dependent RNA polymerase II (RNAPII) transcribes telomeric DNA into telomeric repeat-containing RNA (TERRA). Here, we show that CpG dinucleotide-rich DNA islands, shared among multiple human chromosome ends, promote transcription of TERRA molecules. TERRA promoters sustain cellular expression of reporter genes, are located immediately upstream of TERRA transcription start sites, and are bound by active RNAPII in vivo. Finally, the identified promoter CpG dinucleotides are methylated in vivo, and cytosine methylation negatively regulates TERRA abundance. The existence of subtelomeric promoters, driving TERRA transcription from independent chromosome ends, supports the idea that TERRA exerts fundamental functions in the context of telomere biology.