Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts

Abstract

The intergenic region (IGR) located upstream of the capsid protein gene in dicistroviruses contains an internal ribosome entry site (IRES). Translation initiation mediated by the IRES does not require initiator methionine tRNA. Comparison of the IGRs among dicistroviruses suggested that Taura syndrome virus (TSV) and acute bee paralysis virus have an extra side stem loop in the predicted IRES. We examined whether the side stem is responsible for translation activity mediated by the IGR using constructs with compensatory mutations. In vitro translation analysis showed that TSV has an IGR-IRES that is structurally distinct from those previously described. Because IGR-IRES elements determine the translation initiation site by virtue of their own tertiary structure formation, the discovery of this initiation mechanism suggests the possibility that eukaryotic mRNAs might have more extensive coding regions than previously predicted. To test this hypothesis, we searched full-length cDNA databases and whole genome sequences of eukaryotes using the pattern matching program, Scan For Matches, with parameters that can extract sequences containing secondary structure elements resembling those of IGR-IRES. Our search yielded several sequences, but their predicted secondary structures were suggested to be unstable in comparison to those of dicistroviruses. These results suggest that RNAs structurally similar to dicistroviruses are not common. If some eukaryotic mRNAs are translated independently of an initiator methionine tRNA, their structures are likely to be significantly distinct from those of dicistroviruses.

FIGURE 1.

Internal initiation of translation mediated by the intergenic region of Taura syndrome virus. (A) Structures of plasmids used for in vitro transcription. Triangles indicate T7 promoter sequences. Numbers mark nucleotide positions in the TSV genome sequence. CP indicates the 5′ part of the capsid coding sequence to produce a firefly luciferase (Fluc) fusion protein. (B) Effect of the 5′ cap and cap analogs on translation of transcripts from pIGR-CP-Fluc in vitro. (C) Effect of the 5′ cap and cap analogs on translation from the second cistron mediated by the IGR-sequence of TSV. (D) Compensatory mutations introduced into the predicted extra side stem and PK I. Dots and asterisks indicate base-pair formation for helical segments and PK I. Mutated nucleotides are colored. (E) Detection of translation products from various mutants. Asterisk marks position of endogenous biotinylated protein in wheat germ extracts.

FIGURE 2.

Initial screening of domain 2-like sequences from databases. (A) List of number of nucleotides in each structural unit in domain 2 of IGR-IRES elements of dicistroviruses. The last line in each column represents the minimum and maximum number of nucleotides in the unit in dicistroviruses. Abbreviation of virus names: PSIV, Plautia stali intestine virus; HiPV, Himetobi P virus; TrV, Triatoma virus; DCV, Drosophila C virus; CrPV, Cricket paralysis virus; BQCV, Black queen-cell virus; RhPV, Rhopalosiphum padi virus; ALPV, Aphid lethal paralysis virus; TSV, Taura syndrome virus; ABPV, Acute bee paralysis virus; KBV, Kashmir bee virus. (B) A representative secondary structure model of domain 2 of IGR-IRES in dicistroviruses. Circles and dots indicate nucleotides and base-pair formations, respectively. Characters “p” and “s” denote sequentially designated structural units. Complementary sequences in the model are shown as r1~pn or r2~pn according to permission for nucleotide pairs comprising each helical segment. Green letters indicate nucleotides that are recognized by the 40S ribosome. s5, which is observed in DCV, RhPV, ALPV, and TSV, is shown in halftone. (C) Parameter B used for initial database search. (D) Result of the initial search against databases listed in Table 1 ▶ using parameter B. Extracted sequence files are available from http://cse.nias.affrc.go.jp/nakaji/RNA_F2DF3C.htm.

FIGURE 3.

Detailed structure search against extracted data set in Figure 2D ▶. (A) A representative secondary structure model of IGR-IRES in dicistroviruses. Circles and dots indicate nucleotides and base-pair formations, respectively. Halftones in domain 1 means these regions are not included parameters. The two types of domain 3 are separately shown. Characters “p” denote sequentially designated structural units. Complementary sequences in the model are shown as rn~pn according to permission for nucleotide pairs comprising each helical segment. Green letters indicates conserved loop sequences in dicostroviruses. (B) Parameters used for detailed search. To extract dicistroviral IGR-IRES sequences, parameter A1 was constructed. Three numbers in square brackets represent the number of nucleotides permitted for mismatches, insertions, and deletions, respectively. Parameters A2, A3, A4, and A5 were modified from parameter A1 to loosen the search definitions. The loosened structural units were marked in red and linked to parameter A1 with lines. Parameter D2 extracts sequences similar to domain 2. Parameter D5 is a loosened version of parameter D2. (C) The result of a detailed search against data set extracted in Figure 2D ▶ using parameters shown in (B). Extracted sequence files are available from http://cse.nias.affrc.go.jp/nakaji/RNA_F2DF3C.htm.