310 research outputs found
In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles
The NHPX protein is a nucleolar factor that binds directly to a conserved RNA target sequence found in nucleolar box C/D snoRNAs and in U4 snRNA. Using enhanced yellow fluorescent protein (EYFP)– and enhanced cyan fluorescent protein–NHPX fusions, we show here that NHPX is specifically accumulated in both nucleoli and Cajal bodies (CBs) in vivo. The fusion proteins display identical localization patterns and RNA binding specificities to the endogenous NHPX. Analysis of a HeLa cell line stably expressing EYFP–NHPX showed that the nucleolar accumulation of NHPX was preceded by its transient accumulation in splicing speckles. Only newly expressed NHPX accumulated in speckles, and the nucleolar pool of NHPX did not interchange with the pool in speckles, consistent with a unidirectional pathway. The transient accumulation of NHPX in speckles prior to nucleoli was observed in multiple cell lines, including primary cells that lack CBs. Inhibitor studies indicated that progression of newly expressed NHPX from speckles to nucleoli was dependent on RNA polymerase II transcription, but not on RNA polymerase I activity. The data show a specific temporal pathway involving the sequential and directed accumulation of NHPX in distinct subnuclear compartments, and define a novel mechanism for nucleolar localization
UV-induced fragmentation of Cajal bodies
The morphology and composition of subnuclear organelles, such as Cajal bodies (CBs), nucleoli, and other nuclear bodies, is dynamic and can change in response to a variety of cell stimuli, including stress. We show that UV-C irradiation disrupts CBs and alters the distribution of a specific subset of CB components. The effect of UV-C on CBs differs from previously reported effects of transcription inhibitors. We demonstrate that the mechanism underlying the response of CBs to UV-C is mediated, at least in part, by PA28γ (proteasome activator subunit γ). The presence of PA28γ in coilin-containing complexes is increased by UV-C. Overexpression of PA28γ, in the absence of UV-C treatment, provokes a similar redistribution of the same subset of CB components that respond to UV-C. RNA interference–mediated knockdown of PA28γ attenuates the nuclear disruption caused by UV-C. These data demonstrate that CBs are specific nuclear targets of cellular stress-response pathways and identify PA28γ as a novel regulator of CB integrity
Nuclear processes controlled by molecular machines
A report on the 'Nuclear Structure and Function' symposium at the joint spring meeting of the British Society for Cell Biology, British Society for Developmental Biology and Genetics Society, York, UK, 20-23 March 2002
Cajal bodies and coilin—moving towards function
Many nuclear factors are concentrated within nonmembrane-bound subnuclear bodies. The Cajal body is an example of a conserved nuclear compartment that has been linked to molecular disease. Recent studies have shown Cajal bodies to be surprisingly mobile and offer clues about their function in the cell
Unlocking the chromatin code by deciphering protein–DNA interactions
Characterizing the composition of protein complexes bound to different genomic loci is essential for advancing our mechanistic understanding of transcriptional regulation. In their recent study, Krijgsveld and colleagues (Rafiee et al, 2016) report ChIP‐SICAP, a powerful tool for deciphering the chromatin proteome by combining chromatin immunoprecipitation, selective isolation of chromatin‐associated proteins and mass spectrometry
Spatial mapping of splicing factor complexes involved in exon and intron definition
We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5′ or 3′ splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)–associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains
Signal enhanced proteomics:a biological perspective on dissecting the functional organisation of cell proteomes
Quantitative profiling of the human substantia nigra proteome from laser-capture microdissected FFPE tissue
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