Translation initiation on mammalian mRNAs with structured 5'UTRs requires DExH-box protein DHX29

Abstract

Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA(Met)(i) attach to the 5'-proximal region of mRNA and then scan along the 5' untranslated region (5'UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5'UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.

Figure 1. DHX29 is essential for efficient initiation on mRNAs with structured 5′-UTRs

(A) Left panel - purification scheme for DHX29, right panel - purified DHX29 resolved by SDS-PAGE. (B) Model of the domain organization of DHX29 (upper panel) and alignment of conserved motifs in the helicase core domains of human DHX29 and representative DExH-box proteins (lower panel). (C-E) Toe-printing analysis of 48S complex assembly on (C) CAA-GUS mRNAs containing stems of various stabilities, (D) NCF2 and (E) CDC25 mRNAs. (F) Formation of elongation complexes on CAA-Stem3,4-MVHC-STOP mRNAs assayed by toe-printing (left panel) and by SDG centrifugation with subsequent monitoring of ³⁵S-MVHC tetrapeptide (right panel). P-site mRNA codons and positions of assembled ribosomal complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequences.

Figure 2. DHX29 suppresses the aberrant toe-print +8-9 nt from the AUG codon

Toe-printing analysis of 48S complex assembly on (A, B) β-globin mRNA, (C) mRNA containing two AUG triplets, and (D) CAA-GUS Stem-1 mRNA in RRL (A, lane 3) and in an in vitro reconstituted initiation system (A-D) with eIFs as indicated. Initiation codons and positions of assembled ribosomal complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequences.

Figure 3. Interaction of DHX29 with 40S subunits

Association of DHX29 with (A) individual 40S and 60S subunits, 80S ribosomes, 40S/eIF3/(CUUU)₉ complexes and 43S complexes containing 40S subunits and eIFs 2/3/1/1A, (B) yeast 40S subunits, and (C) 40S/eIF3/(CUUU)₉ complexes in the presence/absence of nucleotides as indicated (lanes 4-7). (D) DHX29 preparation containing a C-terminally truncated fragment resolved by SDS-PAGE (left panel) and its association with 40S subunits (right panel). Ribosomal peak fractions obtained by SDG centrifugation were analyzed by SDS-PAGE and fluorescent SYPRO staining (A-C) and/or western blotting using DHX29 antibodies (A, D). (E) Association of DHX29 with ribosomal complexes in RRL in the presence of GMPPNP assayed by SDG centrifugation. In addition to optical density, the ribosomal profile of RRL was analyzed by scintillation counting to monitor [³⁵S]Met-tRNA^Met_i incorporation. Gradient fractions were analyzed by western blotting using DHX29 antibodies. (F) Estimation of the proportion of 40S-bound DHX29 relative to free protein (upper panel) and of the ratio of DHX29-bound vs. unbound 40S-ribosomal complexes assayed by western blotting using DHX29 antibodies (lower panel).

Figure 4. Ribosomal position of DHX29

Enzymatic (A) and chemical (B) foot-printing analysis of 18S rRNA in 43S and 43S/DHX29 complexes. The positions of residues protected by DHX29 from RNase V1 cleavage and CMCT/DMS modification or cleaved by RNase T1 are indicated. (C, D) 18S rRNA nucleotides protected by DHX29 mapped onto (C) the secondary structure of rabbit 18S rRNA and (D) the crystal structure of the mRNA/T. thermophilus 30S subunit complex (PDB 2HGR). mRNA (blue) and 16S rRNA (grey) are in ribbon representation. Helix 16 is red, protected nucleotides are yellow.

Figure 5. Stimulation of 48S complex formation by DHX29 requires its NTPase activity

(A) Thin-layer chromatography analysis of DHX29′s NTPase activity in the presence/absence of SDG-purified 43S complexes comprising 40S subunits and eIF2/3/1/1A. 10 μl reaction mixtures containing 1 pmol DHX29, 1 pmol 43S complexes and 6.7 μM [α-³²P]ATP, [α-³²P]GTP, [α-³²P]UTP or [α-³²P]CTP, as indicated, were incubated at 37°C for 40 minutes. The positions of [³²P]-NDPs are indicated. (B) Time courses of ATP hydrolysis by DHX29 in the presence/absence of (CUUU)₉ RNA, 18S rRNA, 43S complexes, or 43S/(CUUU)₉, as indicated. 10 μl reaction mixtures containing 0.3 pmol DHX29, 6.7 μM [γ-³²P]ATP and 20 pmol (CUUU)₉ RNA, 0.3 pmol 18S rRNA, 0.3 pmol 43S complexes, or 0.3 pmol 43S complexes with 20 pmol (CUUU)₉ RNA, as indicated, were incubated at 37°C. Aliquots were removed after 2-30 minutes. (C) Toe-printing analysis of 48S complexes assembled on CAA-GUS Stem-1 mRNA in the presence of SDG-purified 43S complexes, DHX29 and NTPs or non-hydrolyzable NTP analogues, as indicated. The position of 48S complexes is indicated.

Figure 6. Helicase activity of DHX29

(A, B, C) Non-denaturing PAGE showing unwinding of (A) 13-bp, (B) 10-bp RNA duplexes with 25 nt-long single-stranded overhanging 5′-regions, and (C) RNA duplexes resembling Stems 2-4 by DHX29, 43S complexes, 43S/DHX29 complexes and eIF4A/eIF4F, as indicated. 1 nM duplex was incubated with 0.15 μM DHX29, 50 nM 43S complexes, 50 nM 43S/DHX29 complexes or 0.15 μM eIF4A/eIF4F and 0.2 mM NTPs, as indicated, at 37°C for 40 minutes. Mobilities of duplex and single-standed RNAs are indicated schematically on the left. 95°C represents the control for denatured strands. (D) SDG-purified 43S complexes containing different amounts of DHX29 and analyzed by SDS-PAGE and fluorescent SYPRO staining. (E, F) Toe-printing analysis of 48S complex formation on CAA-GUS Stem-1 mRNA in the presence of SDG-purified free 43S complexes and 43S complexes containing different amounts of DHX29 (shown in panel D). The positions of the initiation codon and assembled 48S complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequence. (G) Association of DHX29 (and eIF2, as a loading control) with 43S complexes and 48S complexes assembled on native globin mRNA assayed by SDG centrifugation and western blotting.

Figure 7. Influence of DHX29 on 48S complex formation on viral IRESs

Toe-printing analysis of 40S/IRES binary and 48S complexes assembled on (A, B) CrPV, (C) SPV9, (D) wt and ΔDomain II CSFV, and (E) EMCV IRESs in the presence of eIFs as indicated. Initiation codons and positions of assembled ribosomal complexes are indicated. Lanes C/T/A/G depict corresponding DNA sequences.