Speculating on the Roles of Nuclear Speckles: How RNA-Protein Nuclear Assemblies Affect Gene Expression.

Nuclear speckles are eukaryotic nuclear bodies enriched in splicing factors. Their exact purpose has been a matter of debate. The different proposed roles of nuclear speckles are reviewed and an additional layer of function is put forward, suggesting that by accumulating splicing factors within them, nuclear speckles can buffer the nucleoplasmic levels of splicing factors available for splicing and thereby modulate splicing rates. These findings build on the already established model that nuclear speckles function as a storage/recycling site for splicing factors. Many studies have demonstrated proximity between nuclear speckles and sites of active transcription, suggesting that this juxtaposition can enhance the rates of gene expression. It is found that nuclear speckle disassembly increases splicing factor availability in the nucleoplasm, leading to an increase in splicing rates and faster release of nascent transcripts from the gene after transcription. Altogether, this era in which genomic and imaging approaches are applied to study nuclear organization has expanded the outlook on the possible roles of nuclear speckles.

The Association of MEG3 lncRNA with Nuclear Speckles in Living Cells.

Nuclear speckles are nuclear bodies containing RNA-binding proteins as well as RNAs including long non-coding RNAs (lncRNAs). Maternally expressed gene 3 (MEG3) is a nuclear retained lncRNA found to associate with nuclear speckles. To understand the association dynamics of MEG3 lncRNA with nuclear speckles in living cells, we generated a fluorescently tagged MEG3 transcript that could be detected in real time. Under regular conditions, transient association of MEG3 with nuclear speckles was observed, including a nucleoplasmic fraction. Transcription or splicing inactivation conditions, known to affect nuclear speckle structure, showed prominent and increased association of MEG3 lncRNA with the nuclear speckles, specifically forming a ring-like structure around the nuclear speckles. This contrasted with metastasis-associated lung adenocarcinoma (MALAT1) lncRNA that is normally highly associated with nuclear speckles, which was released and dispersed in the nucleoplasm. Under normal conditions, MEG3 dynamically associated with the periphery of the nuclear speckles, but under transcription or splicing inhibition, MEG3 could also enter the center of the nuclear speckle. Altogether, using live-cell imaging approaches, we find that MEG3 lncRNA is a transient resident of nuclear speckles and that its association with this nuclear body is modulated by the levels of transcription and splicing activities in the cell.

Quantifying ß-catenin subcellular dynamics and cyclin D1 mRNA transcription during Wnt signaling in single living cells.

Signal propagation from the cell membrane to a promoter can induce gene expression. To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/ß-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of ß-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear ß-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. ß-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating ß-catenin nuclear dynamics to cyclin D1 transcriptional activation showed that the nuclear accumulation rate of change of the signaling factor, and not actual protein levels, correlated with the transcriptional output of the pathway.