A cell cycle-dependent protein serves as a template-specific translation initiation factor - PubMed (original) (raw)

. 2000 Aug 15;14(16):2028-45.

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A cell cycle-dependent protein serves as a template-specific translation initiation factor

E V Pilipenko et al. Genes Dev. 2000.

Abstract

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) and requires both eukaryotic initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). We show here that the requirements for trans-acting factors differ between related picornavirus IRESs and can account for cell type-specific differences in IRES function. The neurovirulence of Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) was completely attenuated by substituting its IRES by that of foot-and-mouth disease virus (FMDV). Reconstitution of initiation using fully fractionated translation components indicated that 48S complex formation on both IRESs requires eIF2, eIF3, eIF4A, eIF4B, eIF4F, and the pyrimidine tract-binding protein (PTB) but that the FMDV IRES additionally requires ITAF(45), also known as murine proliferation-associated protein (Mpp1), a proliferation-dependent protein that is not expressed in murine brain cells. ITAF(45) did not influence assembly of 48S complexes on the TMEV IRES. Specific binding sites for ITAF(45), PTB, and a complex of the eIF4G and eIF4A subunits of eIF4F were mapped onto the FMDV IRES, and the cooperative function of PTB and ITAF(45) in promoting stable binding of eIF4G/4A to the IRES was characterized by chemical and enzymatic footprinting. Our data indicate that PTB and ITAF(45) act as RNA chaperones that control the functional state of a particular IRES and that their cell-specific distribution may constitute a basis for cell-specific translational control of certain mRNAs.

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Figures

Figure 1

Figure 1

Structures of chimeric Theiler's murine encephalomyelitis virus (TMEV)/foot-and-mouth disease virus (FMDV) RNAs, template activities, and viral phenotypes. (A) Schematic representations of the TMEV IRES showing the positions of the oligopyrimidine tract (Yn) and the initiation codon AUG1068–70 and of constructs derived from the recombinant cDNA for TMEV GDVII (white) and FMDV 01K (black) fused to a luciferase reporter gene (grey). The sizes of cDNA fragments used in these constructs are as indicated; TMEV nucleotide positions (Law and Brown 1990) are shown above pLG, and FMDV nucleotide positions are shown below pLGF-68 and pLGF-80. (B) Template activities of pLG, pLGF-68, and pLGF-80 mRNA transcripts translated in RRL and in BHK-21 cells. (C) In vitro growth characteristics and neurovirulence of wt and chimeric TMEV GDVII viruses. Standard plaque assays were done using virus stocks produced by three accumulating passages posttransfection in BHK-21 cells, and plaques were measured 3 days postinfection. Neurovirulence was assayed by intracerebral inoculation of BALB/c mice with tenfold dilutions of virus. The paralytogenic activity of viruses was expressed as the TCD50 dose causing paralysis in 50% of animals (PD50).

Figure 2

Figure 2

Secondary structures of segments of (A) Theiler's murine encephalomyelitis virus (TMEV) GDVII and (B) foot-and-mouth disease virus (FMDV) 01K internal ribosomal entry sites (IRESs; Pilipenko et al. 1989). The FMDV IRES was linked differently to downstream TMEV sequences in GD68 and GD80 as shown. FMDV and TMEV initiation codons are underlined. Prominent sites of RT arrest caused by eIF4F, PTB, and 48S complexes are indicated by symbols as described in the key at bottom right.

Figure 3

Figure 3

Toeprint analysis of 48S complexes assembled on Theiler's murine encephalomyelitis virus (TMEV) GDVII and foot-and-mouth disease virus (FMDV) 01K internal ribosomal entry sites in RRL. TMEV GDVII mRNA (lane 3) and chimeric GD68 mRNA containing the FMDV IRES (lanes 1,2) were incubated in RRL (lanes 2,3) or with translation components as indicated (lane 1) under standard conditions for 48S complex formation. A primer was annealed to TMEV nt 1195–1214 in the coding region of these mRNAs and extended with AMV-RT. The full-length cDNA extension product is marked E. cDNA products labeled + 17–20 nt terminated at these positions 3′ to the A of TMEV initiation codon AUG1068–70. Reference lanes T, A, C, and G depict the negative-strand sequence derived using the same primer and GD68 plasmid DNA. The junction between TMEV GDVII and FMDV 01K nucleotides is indicated.

Figure 4

Figure 4

Factor dependence of 48S complex formation on the Theiler's murine encephalomyelitis virus (TMEV) internal ribosomal entry site. (A) Toeprinting analysis of RNP and 48S ribosomal complexes assembled on wt TMEV GDVII nt 1–1334 RNA under standard conditions as follows: with aminoacylated initiator tRNA and eIF2, eIF3, eIF4A, and eIF4B (lanes 2–11); with eIF4F (lanes 4–11); with 40S subunits (lanes 3,5,8–11); PTB (lanes 2–5,7,9,11); and ITAF45 (lanes 7,10,11). A primer was annealed within the TMEV coding sequence and was extended with AMV-RT. (B) Toeprinting analysis of the ribonucleoprotein complex assembled by incubating wt TMEV GDVII nt 1–1334 RNA with (lane 2) or without (lane 1) eIF4F. The full-length cDNA extension product is marked E. Other cDNA products terminated at the sites indicated on the right. Reference lanes T, A, C, and G depict the negative-strand TMEV sequence derived using the same primer.

Figure 5

Figure 5

Purification of internal ribosomal entry site (IRES) _trans_-acting factors from rabbit reticulocyte lysate required for internal ribosomal entry on the foot-and-mouth disease virus (FMDV) 01K IRES, using toeprinting to assay 48S complex assembly. GD68 mRNA was incubated under standard reaction conditions for assembly of 48S complexes with translation components as indicated and (A) with the 70%–95% AS fraction (lane 8) or with the 50%–70% AS fraction (lane 9) and with the 100 m

m

KCl DEAE flow-though (lanes 4,7), the 250-m

m

KCl DEAE elution fraction (lanes 5,7), or the 400-m

m

DEAE elution fractions (lanes 6,7) of the RSW 70%–95% AS fraction; (B) with the 400-m

m

KCl phosphocellulose elution fraction (lane 3) or with 185-m

m

KCl (lane 4) or 210-m

m

KCl (lane 5) MonoQ elution fractions derived from this phosphocellulose elution fraction; and (C) with recombinant PTB (lanes 2,4,6) and recombinant ITAF45 (lanes 2,5,6). A primer was annealed to Theiler's murine encephalomyelitis virus (TMEV) nt 1195–1214 in the coding region of GD68 mRNA and was extended with AMV-RT. The full-length cDNA extension product is marked E. cDNA products labeled +17–+20 nt terminated at these positions 3′ to the A of TMEV initiation codon. Other cDNA products terminated at the sites indicated on the right. Reference lanes T, A, C, and G depict the negative-strand sequence derived using the same primer and GD68 plasmid DNA. The junction between TMEV GDVII and FMDV 01K nucleotides and the position of the initiation codon are indicated.

Figure 6

Figure 6

Identical activities of native and recombinant ITAF45 in foot-and-mouth disease virus internal-ribosomal-entry-site-mediated translation initiation. Toeprinting analysis of 48S and RNP complexes assembled on GD80 mRNA under standard conditions for assembly of 48S complexes as follows: with aminoacylated initiator tRNA and eIFs eIF2, eIF3, eIF4A, eIF4B, and eIF4F (lanes 2_–_6) with 40S subunits (lanes 3_–_6), recombinant PTB-1 (lanes 2,4,6) and (A) native ITAF45 (lanes 2,5,6) or (B) recombinant His6–ITAF45 (lanes 2,5,6). A primer was annealed to Theiler's murine encephalomyelitis virus nt 1195–1214 of GD80 mRNA and was extended with AMV-RT. The full-length cDNA extension product is marked E. Other cDNA products terminated at sites indicated on the right of each panel. Reference lanes T, A, C, and G depict the negative-strand sequence derived using the same primer and GD80 plasmid DNA. The position of the initiation codon is indicated.

Figure 7

Figure 7

Purification and structure of ITAF45. (A) Scheme for the purification of ITAF45 from the 0.5

m

KCl RSW from RRL. (B) Resolution by SDS-PAGE of native rabbit ITAF45 (2 μg) after purification by MonoQ chromatography and elution with 210 m

m

KCl. The positions of molecular weight markers are indicated to the left and those of ITAF45 to the right. (C) Schematic illustration of ITAF45 with p67 and MAP to show regions of homology. (D) Deduced amino acid sequence of ITAF45 and alignment of it with sequences of the PAS1 protein from Fugu ribripes (PAS1; Gellner and Brenner 1999), p42 protein from Schizosaccharomyces pombe (p42; Yamada et al. 1994), and with type II methionine aminopeptidases from human (p67; Arfin et al. 1995) and from the hyperthermophile Pyrococcus furiosus (MAP; Tahirov et al. 1998). The sequences of these proteins and ITAF45 show 71%, 26%, 23%, and 35% amino acid identity and 80%, 44%, 43%, and 48% similarity, respectively. Residues in bold are present in the majority of sequences and have been introduced to facilitate visualization of alignments.

Figure 8

Figure 8

Dependence of foot-and-mouth disease virus (FMDV) internal-ribosomal-entry-site (IRES)-mediated internal ribosomal entry on PTB and ITAF45, assayed by sucrose-density gradient centrifugation. Assays were carried out under standard conditions using GD68 or GD80 nt 1–1334 mRNA as indicated. Assembly reactions contained eIFs eIF2, eIF3, eIF4A, eIF4B, and eIF4F, and included recombinant PTB-1 and/or His6–ITAF45, as indicated. Sedimentation was from right to left. Fractions from the upper part of the sucrose gradient were omitted for clarity.

Figure 9

Figure 9

UV cross-linking of ITAF45 to wt Theiler's murine encephalomyelitis virus (TMEV) GDVII, chimeric foot-and-mouth disease virus (FMDV)/TMEV GD80 and β-globin mRNAs. (A) Native and (B) recombinant ITAF45 were UV cross-linked to [32P]UTP-labeled wt TMEV GDVII nt 1–1334, chimeric FMDV/TMEV GD80 nt 1–1334 or β-globin RNA nt 1–600 RNAs in the presence of a 50× molecular weight excess of tRNA and in the presence or absence a 15× molecular weight excess of ribosomal RNA, as indicated.

Figure 10

Figure 10

(A,B) Primer extension analysis of a ribonucleoprotein complex formed on the foot-and-mouth disease virus (FMDV) internal ribosomal entry site (IRES). GD80 mRNA was incubated under standard conditions with ITAF45, PTB, eIF4A, eIF4B, and eIF4G607–1076 individually or in combination as indicated. An oligonucleotide was annealed within the coding sequence and extended with AMV-RT. The cDNA products labeled U664, G665, GU671–2, GCCG681–4, and UUU690–2 terminated at these nucleotides. Reference lanes C, T, A, and G depict the negative strand FMDV sequence.

Figure 11

Figure 11

Chemical and enzymatic footprinting of the foot-and-mouth disease virus (FMDV) internal ribosomal entry site (IRES) in complexes formed with the IRES and ITAF45, PTB, eIF4A, eIF4B, and eIF4G607–1076 individually or in combination as indicated. Polyacrylamide–urea gel fractionation of cDNA products obtained after primer extension showing the sensitivity of the internal ribosomal entry site to RNase T1 cleavage (A, lanes 2–7; B, lanes 1–3; C, lanes 2–5) and to CMCT modification (D, lanes 1,2) either alone or complexed with factors as indicated. cDNA products derived from untreated RNA are shown in lane 1 of panels A and C, in lane 4 of panel B, and in lane 3 of panel D. A dideoxynucleotide sequence generated with the same primer was run in parallel on each gel and is shown in panels A, B, and D. The positions of protected residues or enhanced cleavage are indicated to the right of each panel. The positions of FMDV nucleotides at 50-nt intervals are indicated by black squares to the right of each panel. Individual subdomains are indicated to the left of each panel.

Figure 12

Figure 12

Summary of changes in modification by CMCT and in cleavage by RNAse T1 of the foot-and-mouth disease virus (FMDV) internal ribosomal entry site (IRES) caused by binding of ITAF45, PTB, eIF4A, and eIF4G607–1076 individually or in combination as indicated. These probes are indicated by symbols, as described in the key at top right. Results are displayed on a secondary structure model (Pilipenko et al. 1989). The initiation codon AUG714 is underlined.

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