TERRA transcripts localize at long telomeres to regulate telomerase access to chromosome ends.

The function of TERRA in the regulation of telomerase in human cells is still debated. While TERRA interacts with telomerase, how it regulates telomerase function remains unknown. Here, we show that TERRA colocalizes with the telomerase RNA subunit hTR in the nucleoplasm and at telomeres during different phases of the cell cycle. We report that TERRA transcripts relocate away from chromosome ends during telomere lengthening, leading to a reduced number of telomeric TERRA-hTR molecules and consequent increase in "TERRA-free" telomerase molecules at telomeres. Using live-cell imaging and super-resolution microscopy, we show that upon transcription, TERRA relocates from its telomere of origin to long chromosome ends. Furthermore, TERRA depletion by antisense oligonucleotides promoted hTR localization to telomeres, leading to increased residence time and extended half-life of hTR molecules at telomeres. Overall, our findings indicate that telomeric TERRA transcripts inhibit telomere elongation by telomerase acting in trans, impairing telomerase access to telomeres that are different from their chromosome end of origin.

Nanoscale organization of the endogenous ASC speck.

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to formation of the ASC speck, a supramolecular assembly of the inflammasome adaptor protein ASC. Different models, based on ASC overexpression, have been proposed for the structure of the ASC speck. Using dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT), we visualized the ASC speck structure following NLRP3 inflammasome activation using endogenous ASC expression. A complete structure was only obtainable by labeling with both anti-ASC antibodies and nanobodies. The complex varies in diameter between ∼800 and 1000 nm, and is composed of a dense core with emerging filaments. Dual-color confocal fluorescence microscopy indicated that the ASC speck does not colocalize with the microtubule-organizing center at late time points after Nigericin stimulation. From super-resolution images of whole cells, the ASC specks were sorted into a pseudo-time sequence indicating that they become denser but not larger during formation.

A change in biophysical properties accompanies heterochromatin formation in mouse embryos.

The majority of our genome is composed of repeated DNA sequences that assemble into heterochromatin, a highly compacted structure that constrains their mutational potential. How heterochromatin forms during development and how its structure is maintained are not fully understood. Here, we show that mouse heterochromatin phase-separates after fertilization, during the earliest stages of mammalian embryogenesis. Using high-resolution quantitative imaging and molecular biology approaches, we show that pericentromeric heterochromatin displays properties consistent with a liquid-like state at the two-cell stage, which change at the four-cell stage, when chromocenters mature and heterochromatin becomes silent. Disrupting the condensates results in altered transcript levels of pericentromeric heterochromatin, suggesting a functional role for phase separation in heterochromatin function. Thus, our work shows that mouse heterochromatin forms membrane-less compartments with biophysical properties that change during development and provides new insights into the self-organization of chromatin domains during mammalian embryogenesis.

TERRA stability is regulated by RALY and polyadenylation in a telomere-specific manner.

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres that plays key roles in telomere maintenance. A fraction of TERRA is polyadenylated, and the presence of the poly(A) tail influences TERRA localization and stability. However, the mechanisms of TERRA biogenesis remain mostly elusive. Here, we show that the stability of TERRA transcripts is regulated by the RNA-binding protein associated with lethal yellow mutation (RALY). RALY depletion results in lower TERRA levels, impaired localization of TERRA at telomeres, and ultimately telomere damage. Importantly, we show that TERRA polyadenylation is telomere specific and that RALY preferentially stabilizes non-polyadenylated TERRA transcripts. Finally, we report that TERRA interacts with the poly(A)-binding protein nuclear 1 (PABPN1). Altogether, our results indicate that TERRA stability is regulated by the interplay between RALY and PABPN1, defined by the TERRA polyadenylation state. Our findings also suggest that different telomeres may trigger distinct TERRA-mediated responses.