1. 20S Proteasome Differentially Alters Translation of Different mRNAs via the Cleavage of eIF4F and eIF3 
James M. Baugh, Evgeny V. Pilipenko 
Molecular Cell 
Volume 16, Issue 4, Pages (November 2004)
DOI: /j.molcel

2. Figure 1
Translation Initiation of HAV RNA Is Regulated by Stimulatory and Inhibitory Cellular Activities
(A) Toeprint analysis of 48S complexes formed on HAV RNA in the presence of pure translational components and in the RRL. Reactions were assembled with components as indicated in the tables above the gels. Reference lanes T, A, C, and G show the sequence. The positions of two initiator AUGs are shown to the left, and the positions of cDNA products terminated due to the 48S complex formation are labeled to the right.
(B) Toeprint analysis of 48S complex formation in the presence of postribosomal fractions precipitated with ammonium sulfate (A.S.).
(C) The role of individual eIFs and TRASPs in translation initiation at HAV IRES.
(D) Inhibitory activity exhibits a dominant-negative effect on the 48S complex formation.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
20S Proteasome Is Responsible for the Inhibition of Translation Initiation of HAV RNA
(A) Purification protocol of the inhibitory activity.
(B) Coomassie-stained SDS-PAGE of the purified inhibitory activity (lane 2) and MW marker (lane 1). The position of the extracted bands and the identification of the proteins comprised therein are shown to the right.
(C) Electron micrographs of the purified inhibitory activity stained with 1% uranyl acetate (upper panel) or 1% phosphotungstic acid (lower panel).
(D) Immunoblot analysis of pure 20S proteasomes (lanes 1 and 2) and the RRL (lanes 3 and 4) with the antibody specific for α2 subunit of the proteasome. The amounts of the probes loaded (picomoles and microliters, respectively) are indicated on the top.
(E) Toeprint analysis of 48S complex formation on HAV RNA in the presence of increasing amounts of 20S proteasome.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
The Native 20S Proteasome Endoproteolytically Cleaves the eIF4G Subunit of eIF4F and the eIF3a Subunit of eIF3
(A) Time course of eIF4G cleavage by the native 20S proteasome. eIF4F (6 pmol) was incubated with 20S proteasome (2 pmol) and stained with Blue R following SDS-PAGE. The positions of eIF4F subunits and the cleavage products of eIF4G (CPs) are marked to the left. MW markers are shown to the right. eIF4G subunit of eIF4F is heterogeneous in size because five isoforms of eIF4GI are produced by alternative translation initiation on its mRNA (Bradley et al., 2002).
(B) Comparison between the SDS-activated (lane 2) and the native (lane 3) 20S proteasomes in cleaving the eIF4G subunit of eIF4F. All probes, including eIF4F alone (lane 1), were incubated for 80 min.
(C) N-terminal sequence of 53 kDa cleavage product of rabbit eIF4G and the alignment of rabbit eIF4GI with human eIF4GI and eIF4GII. The predicted cleavage site is marked with the slash. The amino acid numbers are given for the shortest isoform of rabbit eIF4GI (GI:729820) and the longest isoform of human eIF4GI (Bradley et al., 2002).
(D) Dose-dependent inhibition of the eIF4G cleavage by MG132.
(E) Dose-dependent effect of the Protease Inhibitors Cocktail “Complete” on the cleavage of eIF4G. The amount is expressed as the excess over the concentration recommended by manufacturer.
(F) Dose-dependent effect of epoxomicin (lanes 3–5) and YU102 (lanes 6–8) on the cleavage of eIF4G. Lane 2, eIF4F incubated with 20S proteasomes in the absence of inhibitors.
(G) Dose-dependent inhibition of the eIF4G cleavage by the combination of epoxomicin and YU102.
(H) 20S proteasome is responsible for the cleavage of eIF3a in RRL. Lane 1, eIF3 purified from the RRL in the absence of MG132; lane 2, eIF3 purified in the presence of MG132; lane 3, eIF3 (with the intact eIF3a subunit) incubated with the catalytic core for 10 min. The subunits of eIF3 are shown to the left and the cleavage products are marked with thin arrows.
(I) Immunoblot analysis of the time course of eIF3a cleavage. eIF3 (8 pmol) was incubated with 20S proteasome (2 pmol) for 10, 20, and 40 min (lanes 3, 4, and 5, respectively). Lane 1, eIF3 purified in the absence of MG132; lane 2, eIF3 purified in the presence of MG132.
(J) Blue R staining of the same membrane used for the immunoblot analysis in (I).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
20S Proteasome Inhibits the Assembly of 48S Complexes via Its Endoproteolytic Activity in Cleaving eIF4G and eIF3a
(A) MG132 rescues the assembly of 48S complexes on HAV RNA. The reactions were assembled as indicated, with 8 pmol of 20S proteasomes being used.
(B) Toeprint analysis of 48S complex formation on HCV RNA in the presence of increasing amounts of 20S proteasome. The reactions were assembled as indicated.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 20S Proteasome Sequentially Cleaves eIF4G
(A–E) Immunoblot analysis of the time course of eIF4GI cleavage with antibodies specific to different amino acid sequences of eIF4GI. eIF4F (3 pmol) was incubated with 20S proteasome (2 pmol) for 5, 10, and 20 min (lanes 2, 3 and 4, respectively). Lane 1, intact eIF4F; lane 5, 20S proteasome. The positions of cleavage products are marked to the left, and the positions of MW markers are shown to the right. Cleavage product of ∼45 kDa marked with asterisk in (A) likely corresponds to the N-terminal part of the shortest isoform of eIF4GI.
(F) Blue R staining of eIF4G cleavage products when 30 pmol of eIF4F and 4 pmol of 20S proteasome were incubated for 20 and 80 min. The positions of cleavage products are marked with asterisks.
(G) The model of the eIF4GI cleavage. The upper lane schematically represents the structure of the largest eIG4GI isoform. The filled boxes correspond to the binding sites for PABP, eIF4E, eIF4A, eIF3, eIF4A, and Mnk1 in the direction from the N- to C terminus. The positions of epitopes recognized by different antibodies are mapped by asterisks. The cleavage site 1108D/A1109 was determined by Edman sequencing of C-1′ product. Positions of other cleavage sites (CS) are tentative, based on mapping the cleavage products with specific antibodies and the mobility of the products.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Cleavage of eIF3a or eIF4G by the 20S Proteasome Differentially Affects the Assembly of 48S Complexes on Different mRNAs
(A–C) Toeprint analysis of 48S complex formation on the native capped β-globin mRNA (A), HAV RNA (B), and GDVII RNA (C). The reactions were assembled as indicated above the gels, with 2 pmol of 20S proteasomes being used.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Proteasomes Cleave Translation Factors in the Cell, Thereby Regulating the Activity of Different Viral IRESes In Vivo
(A) Immunoblot analysis of Huh-7 lysates with antibodies specific to the eIF3a. Cells were incubated in the presence or in the absence of 2 μM MG132, and harvested at different time points as indicated. Lane 7, eIF3 purified from the RRL in the presence of MG132; lane 8, eIF3 cleaved by the 20S proteasome. The positions of the eIF3a and its cleavage products are marked to the left, and the positions of MW markers are shown to the right.
(B) Immunoblot analysis of Huh-7, BHK-21, and rat skin cells (RSC; ATCC No. CRL-1213) with antibodies specific to the eIF3a.
(C–H) The effect of the MG132 treatment of Huh-7 (C–E) and BHK-21 cells (F–H) on the activity of HAV, GDVII, and HCV IRES elements. IRES activities are represented as means of the Firefly/Renilla ratio and the standard deviations from triplicate experiments. Light, dark, and black bars correspond to untreated and treated with 2 or 4 μM MG132 cells, respectively. Experiments were repeated three times with consistently reproduced results.
Molecular Cell  , DOI: ( /j.molcel )