Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins

TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed: Specialized transcription factories

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories

The proteomes of transcription factories containing RNA polymerases I, II or III

Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo

Nuclear translation

In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription �����factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).</p

Nuclear translation

In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).</p

Most Human Proteins Made in Both Nucleus and Cytoplasm Turn Over within Minutes

<div><p>In bacteria, protein synthesis can be coupled to transcription, but in eukaryotes it is believed to occur solely in the cytoplasm. Using pulses as short as 5 s, we find that three analogues – L-azidohomoalanine, puromycin (detected after attaching fluors using ‘click’ chemistry or immuno-labeling), and amino acids tagged with ‘heavy’ ¹⁵N and ¹³C (detected using secondary ion mass spectrometry) – are incorporated into the nucleus and cytoplasm in a process sensitive to translational inhibitors. The nuclear incorporation represents a significant fraction of the total, and labels in both compartments have half-lives of less than a minute; results are consistent with most newly-made peptides being destroyed soon after they are made. As nascent RNA bearing a premature termination codon (detected by fluorescence <i>in situ</i> hybridization) is also eliminated by a mechanism sensitive to a translational inhibitor, the nuclear turnover of peptides is probably a by-product of proof-reading the RNA for stop codons (a process known as nonsense-mediated decay). We speculate that the apparently-wasteful turnover of this previously-hidden (‘dark-matter’) world of peptide is involved in regulating protein production.</p></div

Puromycin incorporation. HeLa cells were pre-treated with cycloheximide (100 µg/ml; 15 min) to slow ribosomes, pulsed with puromycin (puro; 91 µM; 0–60 s), fixed, puromycylated peptides immuno-labeled with Cy3, DNA stained with DAPI, and images collected.

<p>(<b>A-D</b>) Typical confocal sections through the centre of nuclei. A 5-s pulse gives bright nuclear foci. After 30 s, cytoplasmic and nuclear signals are more similar and diffuse. After 60 s, the peri-nuclear region is the brightest. Bar: 10 µm. (<b>E</b>) Using wide-field images, Cy3 intensities (±SD; <i>n</i> = 20 cells) are expressed relative to the cytoplasmic value after a 60-s pulse.</p

Aha incorporation. HeLa cells were starved of Met (15 and 30 min for 2-min and 5-s pulses, respectively), pulsed ±2 mM Aha, and chased (0–5 min; 0.2 mM Met without Aha).

<p>After fixation and ‘clicking’ on Alexa555, DNA was counterstained with DAPI, images collected using a wide-field microscope, and fluorescence intensities (± SD) in the cytoplasm (<i>cyto</i>) and nucleus (<i>nuc</i>) normalized relative to values in the untreated cytoplasm. *: <i>P</i><0.0001 (Student's two-tailed <i>t</i> test, <i>n</i> = 20 cells). Bars: 10 µm. (<b>A</b>) 2-min Aha pulse. (i,ii) Aha labels both nucleus and cytoplasm, with the nucleus being the brightest. (iii) Pretreatment with anisomycin (aniso; 100 µg/ml; 2 h) reduces signals in both nucleus and cytoplasm. (iv) Slightly more signal is found in the larger area of the cytoplasm. (v) Signals in both compartments disappear during a chase. (<b>B</b>) After a 2-min pulse, signals in both compartments disappear quickly during a chase. Best fits of simple exponential curves to the data are included, but note that we do not know how many kinetic populations there might be (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099346#pone.0099346.s007" target="_blank">Materials and Methods S1</a>). (<b>C</b>) MG132 (100 µg/ml; 2 h) increases signal given by a 2-min pulse. (<b>D</b>) 5-s Aha pulse. (i-iii) Signal is sensitive to anisomycin (aniso; 100 µg/ml; 15 min).</p

Effects of PTCs on nascent <i>Cd2</i> RNA. Cos-7 cells (which encode the SV40 T antigen) were co-transfected with constructs encoding the ‘Tet’ repressor and a test vector (with the ‘Tet’ promoter driving <i>Cd2</i> ± a PTC).

<p>By 24(which also encodes the SV40 <i>ori</i>) replicates to give ∼8,000 mini-chromosomes/cell; the ‘Tet’ promoter is silent and no <i>Cd2</i> RNA is detected. Now doxycycline (10 µM; 45 min) is added, cells fixed 26 h after transfection, intronic <i>Cd2</i> RNA detected by RNA FISH, DNA stained with DAPI, and images collected using a wide-field microscope. (<b>A,B</b>) Two views of one field after co-transfecting the PTC^– vector (only the cell at the top was transfected and expresses intronic <i>Cd2</i> RNA in nuclear foci). Bar: 10 µm. (<b>C</b>) After subtracting background, intensities (± SD) seen in nuclei are expressed relative to the value found in untreated cells transfected with the PTC¯ vector. *: <i>P</i><0.0004 (Student's two-tailed <i>t</i> test, <i>n</i> = 20 cells). (<b>i</b>) A PTC reduces levels of intronic RNA, but cycloheximide (chx; 100 µg/ml; 2 h) more than reverses the effect. (<b>ii</b>) After transfecting the PTC¯ vector, SSA (100 ng/ml; 45 min) reduces levels of intronic RNA, and anisomycin (aniso; 100 µg/ml; 2 h) reverses this effect.</p

A model illustrating how ‘dark-matter’ peptides (green lines i-iii) and the ‘mature’ proteome (iv) arise.

<p>Most initiated ribosomes terminate prematurely (giving i and iii), and some translate to the end of an uORF (giving ii); the resulting peptides are rapidly degraded (half-life <1 min), to give rise to the astonishing turnover seen using short pulses. A minority of ribosomes translate the whole ORF (giving iv); such peptides are the ones detected conventionally using long pulses (they are generally stable and constitute the ‘mature’ proteome). During long pulses, most peptides i-iii are degraded and so are not detected.</p