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, 15 (5), 932-46

Translation Initiation Factors Are Not Required for Dicistroviridae IRES Function in Vivo

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Translation Initiation Factors Are Not Required for Dicistroviridae IRES Function in Vivo

Nilsa Deniz et al. RNA.

Abstract

The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2*GTP*initiator tRNA(met)) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo.

Figures

FIGURE 1.
FIGURE 1.
CrPV IGR IRES functions efficiently when the catalytic or regulatory subunits of eIF2B are mutated. (A) Diagram of the LEU2 IGR URA3 dicistronic reporter; the CUP1 promoter that drives transcription of the reporter is indicated by an arrow. (B) Serial dilutions of wild-type (GCD7 or GCD6) and mutant (gcd7-201 or gcd6-1) eIF2B yeast strains on SD complete media demonstrate relative growth phenotypes. (C) Growth assays on SD media with 100 μM CuSO4, supplemented without (left) or with (right) uracil. (D) Western analysis of Flag-tagged Ura3 protein (above) expressed from the dicistronic reporter in gcd6-1, GCD6 (left) and gcd7-201, GCD7 (right) yeast strains. The blot was stripped and reprobed with anti-PGK antibody as a loading control (bottom). (E) Northern analysis of total RNA using a probe to the 5′end of the coding region of URA3 gene (top). An arrow indicates the full-length dicistronic message. Asterisk indicates a shorter transcript. The ethidium bromide staining of the 25S rRNA serves as a loading control (bottom).
FIGURE 2.
FIGURE 2.
An eIF3 mutation enhances IGR IRES activity. (A) Growth assay on complete SD media shows relative growth phenotypes of wild-type (PRT1) vs. mutant (prt1-1) eIF3 strains. (B) Growth assay on SD media with 100 μM CuSO4 and supplemented without (left) or with (right) uracil. (C) Immunoblot using anti-Flag M2 monoclonal antibody to detect the C-terminal Flag tag of the Ura3 protein (top). The blot was stripped and reprobed with anti-PGK antibody as a loading control (bottom). (D) Northern analysis of total RNA using 32P labeled DNA probe for the 5′end of the URA3 coding region (top). The ethidium bromide staining of the 25S rRNA serves as a loading control (bottom).
FIGURE 3.
FIGURE 3.
CrPV IGR IRES functions when eIF4G levels are reduced. (A) Growth assay on complete SD media shows relative growth phenotypes of wild-type (TIF4631) vs. mutant (tif4631) eIF4G1 strains. (B) Growth assay on SD media with 100 μM CuSO4 supplemented either without (left) or with (right) uracil. (C) Immunoblot using anti-Flag M2 monoclonal antibodies to detect the C-terminal Flag tagged Ura3 protein (top). The blot was stripped and reprobed with anti-PGK antibody as a loading control (bottom). (D) Northern analysis of total RNA using 32P labeled DNA probe for the 5′end of the URA3 coding region (top). The ethidium bromide staining of the 25S rRNA serves as a loading control (bottom).
FIGURE 4.
FIGURE 4.
The IGR IRES can sustain growth in an eIF4E-ts mutant. (A) Growth assay on complete SD media shows relative growth phenotypes of wild-type (CDC33) and cdc33-4-2 mutant (cdc33-ts) eIF4E strains. (B) Growth assay on SD media with 100 μM CuSO4 without (left) or with (right) uracil. Strains were grown at 27.5°C. (C) Immunoblot using anti-Flag M2 monoclonal antibody to detect the C-terminal Flag tagged Ura3 protein (top). The blot was stripped and reprobed with anti-PGK antibody as a loading control (bottom). (D) Northern analysis of total RNA using 32P labeled DNA probe for the 5′end of the URA3 coding region (top). Ethidium bromide staining of the 25S rRNA serves as a loading control (bottom).
FIGURE 5.
FIGURE 5.
CrPV IGR IRES does not require eIF5B in vivo. (A) Growth assay on complete SD media shows relative growth phenotypes of wild type (FUN12) and a mutant strain lacking eIF5B (fun12Δ). (B) Growth assay on SD media with 100 μM CuSO4 supplemented either without (left) or with (right) uracil. (C) Immunoblot using anti-Flag M2 monoclonal antibodies to detect the C-terminal Flag tagged Ura3 protein (top). The blot was stripped and reprobed with anti-PGK antibody as a loading control (bottom). (D) Northern analysis of total RNA using 32P labeled DNA probe for the 5′end of the URA3 coding region (top). The ethidium bromide staining of the 25S rRNA serves as a loading control (bottom).
FIGURE 6.
FIGURE 6.
An excess of 40S over 60S subunits is not sufficient to enhance IGR IRES function. (A) Growth assay on complete SD media showing relative growth phenotypes of wild-type (RPL16B) and ribosomal mutant (rpl16bΔ) strains. (B) Growth assay on SD media with 100 μM CuSO4 and either without (left) or with (right) uracil. (C) Immunoblot using anti-Flag M2 monoclonal antibodies to detect the C-terminal Flag tagged Ura3 protein (top). The fun12Δ yeast strain with the IGR dicistronic reporter is shown as a positive control. The blot was stripped and reprobed with anti-PGK antibody as a loading control (bottom). (D) Northern analysis of total RNA using 32P labeled DNA probe for the 5′end of the URA3 coding region (top). The ethidium bromide staining of the 25S rRNA serves as a loading control (bottom). (E) Luciferase assays of RPL16B or rpl16bΔ harboring a dicistronic luciferase reporter with either a wild-type or mutant IGR IRES.
FIGURE 7.
FIGURE 7.
In vivo IGR IRES activity is independent of any contributions by a cap-dependent mechanism of translation. (A) Northern analyses of the dicistronic reporter transcripts from gcd7-201 and GCD7 yeast strains using a 32P-labeled oligo probe against the core region (SL2.1) of the CrPV IGR IRES (top). The ethidium bromide staining of the 25S rRNA serves as a loading control (bottom). (B) A diagram of the ΔAUG dicistronic luciferase reporter. (C) Wild-type, tif4631Δ, and fun12Δ yeast strains harboring either the IGR or IGRmut IRES ΔAUG dicistronic luciferase reporter. Graph illustrating the firefly luciferase activity standard error is indicated for n = 3.

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