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The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors

Mark P. Sowden1, Nazzareno Ballatori2, Karen L. de Mesy Jensen3, Lakesha Hamilton Reed* and Harold C. Smith1,2,3,4,{ddagger}

1 From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
3 From the Department of Pathology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
2 From the Department of the Environmental Health Sciences, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
4 From the Department of Cancer Centers, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA
* Present address: Prairie View A & M University, Prairie View, TX 77446, USA



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  		Fig. 1. Amino acid sequence of rat p66 and its alignment with huACF. P66 cDNA was amplified from total rat liver poly A mRNA. The predicted amino acid sequence has been aligned with that of huACF. For clarity, only those amino acids not identical to huACF are shown, with shading indicating a conservative substitution in the rat p66 sequence. The location of the RNP2 and RNP1 sequences of the three RRMs are indicated. The site of an eight amino acid insertion found in the human ASP homolog and the location of the 15 amino acid sequence used to produce peptide-specific antibodies are also indicated.

 


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  		Fig. 2. Rat p66 mooring-sequence-selective, RNA-binding protein is a huACF homolog. Rat liver nuclear 27S editosome glycerol gradient fraction was UV cross-linked to radiolabeled apoB RNA. The proteins in the reaction were resolved by SDS PAGE, immunoblotted and exposed to X-ray film to determine the migration of the known 66 kDa and 44 kDa apoB RNA-binding proteins (left lane). Subsequently, the blot was reacted with anti-ACF (right lane). The positions of p66 and p44 UV cross-linked proteins and the cross-immunoreactive p66 are indicated. The stacking/running gel interface (S/R) is also indicated for reference.

 


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  		Fig. 3. Anti-huACF immunoprecipitation of rat p66 cross-linked to apoB RNA. (A) In vitro editing reactions containing rat liver nuclear extract and either radiolabeled apoB or control RNA (WT-1) were immunoprecipitated with anti-huACF at varying times during a 60 minute reaction and without RNase digest, subjected to immunoprecipitation with anti-huACF antibody bound to Protein A beads. The amount of RNA recovered in the immunoprecipitate was determined by scintillation counting washed beads. (B) In vitro editing reactions containing recombinant huACF or rat liver nuclear extract and radiolabeled apoB RNA, together with 1,000-fold molar excess of either WT-1 or apoB RNA (as indicated above each lane) were UV cross-linked after 60 minutes of reaction, RNase digested and then subjected to immunoprecipitation with anti-huACF antibodies. Immunoprecipitates were resolved on 10% SDS PAGE and autoradiographed.

 


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  		Fig. 4. Expression of P66/ACF mRNA and protein. (A) A northern blot of total cellular rat polyA+ mRNA from the indicated tissues was probed with p66/ACF cDNA and autoradiographed. The relative migration of size markers is shown to the left. (B) A western blot of total cellular protein from the indicated cell lines was reacted with peptide-specific polyclonal antibodies and visualized by chemiluminescence and autoradiography.

 


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  		Fig. 5. Recovery of p66/ACF during rat liver nuclear and cytoplasmic fractionation. (A) Normal rat liver nuclear and cytoplasmic S100 extracts were prepared and subjected to an in vitro editing reaction and poisoned primer extension quantification of editing. RNA editing was quantified by PhosphorImager densitometry as the number of counts in edited RNA (UAA) divided by the sum of the counts in UAA and the unedited RNA (CAA) times 100. The reactions were performed in triplicate and shown with the s.e.m. (B) 25 µg of protein from nuclear (N) and cytoplasmic (C) S100 extracts were resolved by SDS PAGE and stained with Coomassie blue (left pair of lanes) or western blotted with anti-ACF as described in Materials and Methods (right pair of lanes). The migration of molecular mass marker proteins is shown to the left (Mr). (C) Chemiluminescence from western blots in B were quantified, setting the density in the cytoplasmic lane to an arbitrary unit of 1 (first column). The total amount of protein in each fraction was calculated (second column) and this value was multiplied by the relevant number in column 1 to give the estimated amount of total p66/ACF in each fraction (column three). The relative percent p66/ACF in each fractions (column four) was calculated assuming that the sum of the nuclear and cytoplasmic p66/ACF equalled the total tissue p66/ACF. On average, 35 ml of cytoplasmic S100 extract at 39 mg protein/ml and 2 ml of nuclear S100 extract at 21 mg protein/ml were obtained.

 


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  		Fig. 6. Native p66/ACF is distributed in both the nucleus and cytoplasm of McArdle rat hepatoma cells. McArdle cells were grown on glass slides and prepared for immunofluorescence microscopy using anti-ACF. Two different fields are shown, with DAPI nuclear staining (left) and FITC fluorescence of anti-ACF reactivity (right).

 


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  		Fig. 7. Immunohistochemical localization of rat liver p66/ACF. Rat liver sections were prepared and subjected to immunocytochemistry using anti-p66/ACF. (A) Low power (20x) image of a liver section showing the distribution of central veins with associated dense p66 reactivity. (B) Higher power (40x) images of two of the central veins regions in A showing regionally high concentrations of p66/ACF reactivity. Individual hepatocytes were made apparent by nuclear staining with hematoxylin counter staining.

 


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  		Fig. 8. Localization of rat liver p66/ACF by immunoelectron microscopy. Rat liver sections were prepared, reacted with anti-p66/ACF and detected with biotin secondary antibody and peroxidase conjugated extrAvidin, followed by enhancement with silver and gold toning. (A) Magnification, 7500x. (B) High magnification (22,500x) of the 3 o'clock area of the nucleus in A. ER, endoplasmic reticulum, Mt, mitochondria; No, nucleolus; Nu, nucleus.

 


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  		Fig. 9. Ultrastructural localization of p66/ACF. Sections of rat liver were prepared and reacted with p66/ACF as described in Fig. 8. (A) P66/ACF was associated with the surface and borders of heterochromatin in the nucleus (magnification, 22,500x). Cytoplasmic p66/ACF was predominantly associated with the outer surface of the endoplasmic reticulum and B, to a lesser extent with the outer surface of the Golgi (magnification, 30,000x). ER, endoplasmic reticulum; Mt, mitochondria; Nu, nucleus.

 


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  		Fig. 10. Cytoplasmic and nuclear complexes containing editing factors. Cytoplasmic and nuclear S100 extracts were sedimented through 10%-50% glycerol gradients, fractionated and assayed for p66/ACF and KSRP by western blotting or for co-sedimenting APOBEC-1 by assaying in vitro editing activity. Glycerol gradients were loaded with 60 and 20 mg of cytoplasmic and nuclear S100 extracts, respectively. Gradient fractions from cytoplasmic (A,C,E) or nuclear (B,D,F) S100 extracts were analyzed by western blotting. Fractions are numbered from the top of each gradient and the gradient positions corresponding to 11S, 27S and 60S complexes are indicated. An equal aliquot of each gradient fractions was resolved by SDS PAGE and blotted. A and B are blots reacted with antibodies specific for KSRP; blots C and D were reacted with antibodies against p66/ACF. Poisoned primer extension and gel analysis of in vitro editing activity in gradient fractions from cytoplasmic (E) and nuclear (F) S100 extracts correspond to those immunoblotted in panels A/C and B/D, respectively. The primer extension products from unedited (CAA) and edited (UAA) RNA are indicated. The percent editing in each fraction was determined as described in Fig. 5 and was 2%, 2%, 16%, 14% and 14% for fractions 5-9, respectively, in E, and 15%, 15%, 17%, 4% and 0.5% in fractions 4-8 in F, respectively.

 


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  		Fig. 11. Editing activity and the proportion of total cellular p66/ACF in the nucleus are enhanced following ethanol and insulin treatment. RNA was extracted from cultures of rat primary hepatocytes treated with ethanol or insulin for 6 hours. Editing activity was quantified by the poisoned primer extension assay, whereas cultures treated in parallel were subfractionated into cytoplasm proteins and nuclear proteins and assayed for p66/ACF by western blotting. Editing activity is shown as the average of three experiments ± s.e.m. The relevant regions of western blots for cytoplasmic proteins (C) and nuclear proteins (N) are shown and were prepared as described in Fig. 5. The nuclear to cytoplasmic (N/C) ratio was determined as described in Fig. 5 and did not vary more than 10% within treatment groups in the three replicate experiments.

 

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