The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability - PubMed (original) (raw)
The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability
Rob D Chapman et al. Nucleic Acids Res. 2004.
Abstract
The phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD) has been shown to affect the initiation, and transition to elongation of the Pol II complex. The differential phosphorylation of serines within this domain coincides with the recruitment of factors important for pre-mRNA processing and transcriptional elongation. A role for tyrosine and threonine phosphorylation has yet to be described. The discovery of kinases that express a preference for specific residues within this sequence suggests a mechanism for the controlled recruitment and displacement of CTD-interacting partners during the transcription cycle. The last CTD repeat (CTD52) contains unique interaction sites for the only known CTD tyrosine kinases, Abl1/c-Abl and Abl2/Arg, and the serine/threonine kinase casein kinase II (CKII). Here, we show that removal or severe disruption of the last CTD repeat, but not point mutation of its CKII sites, results in its proteolytic degradation to the Pol IIb form in vivo, but does not appear to affect the specific transcription of genes. These results suggest a possible mechanism of transcription control through the proteolytic removal of the Pol II CTD.
Figures
Figure 1
Establishment of cell lines conditionally expressing RNA Pol II LS* mutants. (A) A schematic drawing of the constructs used in this study. A construct containing the HA-tagged, α-amanitin-resistant large subunit of Pol II (LS*wt) was modified to include a multiple cloning site directly before the stop codon of the wild-type sequence (LS*_wt_MCS), allowing the insertion of the cDNA for EGFP in-frame with that of the wild-type Pol II sequence (LS*_wt_EGFP). Deletion mutants were produced by truncation of the CTD to 49 repeats before the addition of either the 50th (LS* 49+50) or 52nd repeat (LS*49+52). (B) Comparison of the amino acid sequence of the different mutants: Addition of an MCS results in the addition of a further seven amino acids to the wild-type CTD; mutants containing the same number of total repeats, but ending in repeat 50 or repeat 52; addition of EGFP creates a large C-terminal extension to the wild-type sequence. (C) A chemical ‘knock-in, knock-out’ system for the analysis of mutant Pol II LS*: expression of the α-amanitin-resistant polymerase is induced by removal of Tc from the cell culture medium; the endogenous Pol II is inhibited through the addition of α-amanatin 24 h following the induction of expression; localization was assessed a further 48 h later. (D) The expression and localization of an EGFP-tagged polymerase (LS*_wt_EGFP) in Raji cells examined using a fluorescence imaging system.
Figure 2
A time course of cell viability in Raji cells expressing LS*mock, LS*49+50, LS*49+52, LS*_wt_MCS and LS*_wt_EGFP. α-Amanitin was added 24 h following the removal of Tc. The number of living (_N_l) and dead cells (_N_d) was determined by trypan blue staining. The percentage of viable cells (V) was calculated using the formula V = 100 × _N_l/(_N_l + _N_d). The re-addition of Tc to a recovering cell line at day 34 (LS*_wt_EGFP + Tc) controls that the resistance to α-amanitin is dependent on the expression of the recombinant Pol II LS*. A representative example of several experiments is shown.
Figure 3
Run-on analysis of transcriptionally engaged Pol II in Raji cells expressing Pol II mutants LS*49+50 or LS*49+52. Radioactively labelled RNA from nuclear run-on reactions was hybridized to ATLAS human 1.2 arrays (Clontech). Of the six fields (A–F), two representative fields (D and E) are shown for each mutant. Cells were grown in the presence of α-amanitin 24 h prior to harvesting. The nuclei from cells additionally exposed to 12 Gy of γ-radiation (+IR) are compared with untreated cells (–IR). Nuclei were harvested 1 h post-irradiation. Experiments were performed with equal amounts of nuclei. A representative example of several experiments is shown.
Figure 4
Appearance of the Pol IIb in cell lines expressing a mutant lacking CTD52. (A) RIPA extracts of stably transfected cell lines grown in the presence or absence of Tc for 24 h were examined following western blot using anti-HA antibody (3F10). The asterisk denotes a non-specific background band, which also demonstrates equal loading. Ψ signifies a degradation product of Pol II LS* other than the IIb form. (B) Expression profiling of mutants containing or lacking the last CTD repeat in the BL cell lines BL29 and Elijah, and the LCL Rosi.
Figure 5
CTD52 is phosphorylated by CKII in vivo. (A) Polyclonal antibodies were raised against peptides encoding CTD52 (DEEN) and a phosphorylated version thereof (DEEP). The monoclonal antibody 8WG16 recognizes the CTD consensus sequence, YSPTSPS. (B) DEEN and DEEP antibodies discriminate between non-phosphorylated and CKII-phosphorylated CTD: CKII-treated and non-treated GST–CTD were examined following western blot for their reactivity against DEEN and DEEP antibodies. The reactivity with 8WG16 is included as a loading control. (C) The immunoreactivity of Pol II from HeLa cells following DRB, actinomycin D (ActD) or heat shock (HS) treatment (∅ = non-treated control). The monoclonal antibody POL 3/3 recognizes all forms of the Pol II LS since it recognizes an epitope outside of the CTD. (D) Treatment with alkaline phosphatase regenerates DEEN activity: cytosolic (Cytosol-X) and nuclear (Nuclear-X) HeLa cell extracts were treated with increasing amounts of alkaline phosphatase, and their reactivity, along with that of whole extract (Whole-X), against DEEN and POL 3/3 antibodies was compared following western blotting.
Figure 6
Expression profiling of Pol II LS mutants. (A) The amino acid sequence of the last CTD repeat is shown for the mutants produced in this study. LS*_wt_MCS consists of the wild-type 52 repeats plus an additional seven amino acids resulting from the introduction of a multiple cloning site in the DNA sequence (MCS). Green boxes signify regions containing the CKII consensus recognition sequence S/TxxD/E. LS*49+52 consists of a total of 50 repeats, whereby the last repeat corresponds to the sequence of CTD52. Variations of this mutant were produced where one or both potential sites for phosphorylation by CKII are mutated (S→A; labelled red). Similarly, mutants LS*49+NS and LS*49+ATM contain a total of 50 repeats, where the last repeat is either a scrambled CTD52 (NS) or consists of repeat 50 of the wild-type sequence plus the c-Abl interaction domain of ATM. In addition, a mutant truncated to the 50th repeat (LS*49+50) was also produced. (B) Cell lines were cultivated in the absence of Tc for 24 h before the addition of α-amanitin and harvesting a further 24 h later. The induction of the recombinant large subunits was analysed by western blot using HA-specific antibodies (3F10). Samples were prepared using Laemmli buffer. The same blot was stripped and re-probed using CTD-specific antibodies (8WG16). The same samples were also screened using antibodies directed against CKII-phosphorylated CTD (DEEP).
References
- Allison L.A., Wong,J.K., Fitzpatrick,V.D., Moyle,M. and Ingles,C.J. (1988) The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster and mammals: a conserved structure with an essential function. Mol. Cell. Biol., 8, 321–329. - PMC - PubMed
- Pinna L.A. (1990) Casein kinase 2: an ‘eminence grise’ in cellular regulation? Biochim. Biophys. Acta, 1054, 267–284. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Miscellaneous