QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 February 2004
doi: 10.1242/jcs.00933

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Iborra, F. J. Articles by Cook, P. R. Search for Related Content PubMed PubMed Citation Articles by Iborra, F. J. Articles by Cook, P. R. Social Bookmarking                         What's this?

Molecular cross-talk between the transcription, translation, and nonsense-mediated decay machineries

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

View larger version (38K): [in a new window]   Fig. 1. Immunofluorescence reveals components involved in translation and NMD are found near nascent transcripts in HeLa nuclei. (A) Some UPF1, 2 and 3 is nuclear. Cells were fixed, UPF1, 2, or 3 indirectly immunolabelled with Cy3, and equatorial sections through nuclei collected using a confocal microscope. (Rows 1-3) Fraction cellular intensity found in nuclei for cells grown with or without leptomycin B or actinomycin D. *Difference relative to value in row 1, significant at 99.999% level (Student's t-test). Although the nuclear signal given by UPF1 and 2 is faint, quantitative analysis reveals it constitutes a significant fraction of the total. Scale bar: 10 µm. (B) Anti-Br binds to Br-RNA (left), but not when blocked by anti-X bound to its target (right). (C) Some antibodies block access of anti-Br to nascent Br-RNA. HeLa cells were grown briefly in Br-U to label nascent transcripts, and fixed; then the resulting nascent Br-RNA was indirectly immunolabelled with Cy3, and single equatorial optical sections through nuclei collected using a confocal microscope. In the absence of a blocking antibody, nuclear fluorescence marks nascent Br-RNA (left), which is reduced by co-incubation with anti-UPF2 (right); other antibodies have intermediate effects (middle). Scale bar: 20 µm. (D) Colocalization revealed by antibody blocking: various antibodies prevent access of anti-Br to Br-RNA (left), and vice versa (right). Fluorescence intensities over >300 nuclei in images like those in the right-hand panel in (C) were expressed relative to those in the left-hand panel. *Difference relative to value in row 1, significant at 99.999% level (Student's t test).

View larger version (52K): [in a new window]   Fig. 2. Interactions between the different machineries. Various antibodies (A-C) or proteins (D) bound to beads were incubated with HeLa nuclear extract, and proteins immunoprecipitating (IP) with bound antibodies or proteins analysed by immunoblotting. aNormal mouse serum; b1/5 extract used for IP applied directly to lane; cbeads coated with anti-biotin, and extract from permeabilized cells containing nascent peptides tagged without (`control') and with biotin (`bio-peptide'); d1/10 extract used for IP applied directly to lane; ebeads coated with anti-Br, and extract from permeabilized cells containing nascent transcripts tagged without (`control') and with bromine (`Br-RNA'); fextract from permeabilized cells containing nascent Br-RNA. (A) CTDP co-immunoprecipitates with another subunit of the polymerase (RPB8), translation initiation factors (left), NMD proteins (middle) and nascent biotin-peptides (right). (B) CTDP and NMD proteins co-immunoprecipitate with proteins in ribosomes and newly made transcripts, but not histone H4. (C) Components involved in NMD (left) and translation (right) co-imunoprecipitate with nascent Br-RNA and RPB8. (D) The CTD interacts with S6 and UPF1, but not VP16.

View larger version (33K): [in a new window]   Fig. 3. Association detected by co-purification. (A) Immunoblot (using antibody directed against N terminus of the largest subunit) showing form IIA and IIO content in fractions released from HeLa cells treated successively with a hypotonic buffer (`cytosol', lane 1), and 0.035, 0.14, and 0.65 M (NH4)2SO4 (`nuclear', lanes 2-4) and RNase A (`RNase', lane 5). After RNase treatment the fraction contains an especially hyper-phosphorylated form (IIO*). Polymerizing activities (pmol UTP incorporated/min/µg protein) sensitive to -amanitin were: 2.7 [polymerase purified by a conventional procedure (Maldonado et al., 1996)], 0.3 (cytosol), 0.4 (0.035 M), 0.2 (0.14 M), 0.3 (0.65 M), 3.5 (sub-nuclear) and 0.1 (negative control lacking DNA). (B) Immunoblots of the RNase-treated fraction before (`input' with 1x and 1/3x loadings in lanes 1 and 2) and after immunopurification using either antibodies directed against the N terminus of the largest catalytic subunit (`anti-pol', lane 3) or normal mouse serum (lane 4). eIF4E, S6, UPF1 and UPF2 co-immunopurify with the forms IIA and IIO (pol) and the CTDP; in contrast, XPB, cdk7, and cdk8 do not.

View larger version (42K): [in a new window]   Fig. 4. The effects of inhibitors on CD2 levels in the nucleus and cytoplasm. Cos-1 cells were transfected with plasmids encoding a fluorescent control protein (EYFP-Mito, an enhanced yellow fluorescent protein fused with a mitochondrial targeting sequence from subunit VIII of cytochrome c oxidase) with or without rat CD2, grown for 24 hours, and treated with or without 20 µM lactacystin for 15 minutes. After fixation, CD2 was indirectly immunolabelled with Cy3 (red), and an equatorial (confocal) section through nuclei collected. Only Cy3 fluorescence is shown. In some cases, 100 µM DRB or 5 µg/ml actinomycin D (act D) was added 15 minutes before lactacystin. (A) Cell transfected with plasmids encoding only the control protein (lines mark cell and nuclear peripheries). It contains intensely fluorescing mitochondria (not visible), and no CD2 (average nuclear and cytoplasmic intensities of 0.009 and 0.04 arbitrary units/pixel respectively). (B) Cell transfected with plasmids encoding CD2 (appears white) and the control protein (yellow, not visible); most CD2 is in the ER, however, some faint fluorescence is found in nuclei, and quantitative analysis shows this to have 9x the intensity of that seen in an equivalent area in A. 24% (untransfected) cells in this population expressed no EYFP-Mito or CD2 (i.e., with CD2 labelling like that in A); this population was not analysed further. Scale bar: 10 µm. (C) Intensities in individual cells. Each panel contains results from >75 cells transfected with CD2, each point indicates intensity (arbitrary units/pixel) in nuclear and cytoplasmic areas of one cell, and each arrowhead the average intensity.

View larger version (90K): [in a new window]   Fig. 5. Instability of newly made nuclear peptides. HeLa cells were permeabilized, allowed to extend nascent peptides in BODIPY-lys-tRNA for 10 minutes in 10 µg/ml MG-132, washed, reincubated in 1 mg/ml cycloheximide for various times, fixed, and confocal sections through the centre of nuclei collected. (A) Cell after initial pulse in BODIPY-lys-tRNA. (B) As A, but after 2 hours chase. Scale bar: 10 µm. (C) Fall in intensity of nuclear and cytoplasmic regions of >50 cells after chases of different times. Lines fit exponential decays; t1/2 of 120 (nucleus) and 690 (cytoplasm) minutes.

View larger version (24K): [in a new window]   Fig. 6. A model for transcript production. (A) The CTD has the potential to associate with sites involved in capping (Fong and Bentley, 2001), transcript degradation (Andrulis et al., 2002; Libri et al., 2002; Lykke-Andersen et al., 2002), translational proofreading (Iborra et al., 2001; Wilkinson and Shyu, 2002), proteolysis (Iborra et al., 2001), splicing and polyadenylation (labelled A) (Maniatis and Reed, 2002). (B) Transcription began as the template bound to the polymerizing complex and was reeled in as the transcript was extruded; the CTD is now hyper-phosphorylated, and a cap has been added. (C) The transcript continues to be extruded through a splicing site as the ribosome/NMD machinery begins proofreading the now-spliced message (and so does not read introns that may contain many termination codons). (D) Once introns are removed (lariat), the transcript is cleaved, poly-adenylated, and exported to the cytoplasm; but if errors are detected, the faulty transcript and peptide produced during proofreading are degraded by nucleases and proteasomes.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter