Fig 1.
Structure of OsMac1 gene and the effect of the UTRc sp38 element for the translation.
(A) Structure of splicing variants of the OsMac1 mRNA. Boxes show the exons. Regions corresponding to the 5’ UTR are indicated by filled boxes. Spliced regions are indicated by V-shaped lines. Differences among UTRa, UTRb, and UTRc are highlighted. "sp38" is shown as an orange-colored box. (B) Reporter assay of the UTRs. Protoplast of the rice suspension-cultured cells were transformed with the reporter gene containing UTRa, UTRb, and UTRc variants placed upstream of the GUS ORF (left panel). As the control, the region of untranslated sequence of 35S–GUS configuration derived from pBI121 (“no UTR”) was used. After transfection, the cells were incubated for 18 h, and GUS activities were determined. Right panel indicates the relative GUS activities normalized against the GUS mRNA, whose amount was estimated by real-time qRT–PCR. The value of the GUS activity with UTRc was set as 1.0. The nucleotide sequence of the additional 31 nt is shown in S1 Fig. The results represent the means of three independent experiments. Error bars indicate the SD (n = 3). Asterisks indicate significant differences in the translational efficiency of UTRc at P < 0.05.
Fig 2.
Translational efficiency of the UTRc containing modified sp38 regions.
Regions of substitution in sp38 are indicated by shaded boxes (left panel). Numbers in the names of mutant genes indicate the regions of nucleotides that are substituted by the complementary ones. Right panel indicates the relative GUS activities normalized against the GUS mRNA, whose amount was estimated by real-time qRT–PCR. The value of the GUS activity with the wild-type UTRc was set as 1.0. The results represent the means of three independent experiments. Error bars indicate the SD (n = 3).
Fig 3.
Examination of the IRES function in UTRc.
"no UTR" and "UTRc" indicate 35S–GUS and UTRc–GUS, respectively. "ORFVII-UTRc" indicates the ORFVII–UTRc–GUS reporter that contains CaMV ORFVII upstream of the UTRc sequence. The results are expressed as the ratio of the enzymatic activity of GUS and the amount of corresponding GUS-containing mRNA synthesized in cultured rice cells, where the value of the GUS activity expressed from monocistronic mRNA is set as 1.0. GUS mRNA levels were estimated by real-time qRT–PCR. Error bars indicate the SD (n = 3). Asterisks indicate significant differences in the translational efficiency of UTRc at P < 0.05. The results shown represent the means obtained in three independent experiments.
Fig 4.
Identification of regulatory UTRc cis-elements involved in the modulation of OsMac1 mRNA translation.
(A) Mutations introduced in the UTRc leader to exclude uORF1, uORF2, uORF3 or all the AUG codons (AUG codon was replaced by UUG). UTRc-, UTRc (ΔuORF1)-, UTRc (ΔuORF2)-, UTRc (ΔuORF3)-, and UTRc (ΔAlluORF)-containing reporters were used for transformations. (B) Translational efficiency of truncated UTRc mutants. UTRc (Δ100), UTRc (Δ200), UTRc (Δ252), and UTRc (Δ306) are truncated UTRc mutants in which regions of 100 nt, 200 nt, 252 nt and 306 nt were deleted from the 5’ end, respectively, as shown by broken lines. sp38 and uORFs are shown as orange-colored boxes and enlarged open boxes, respectively. Translational efficiencies are shown as the relative GUS activities that are normalized by the amount of mRNA. The value of the GUS activity of UTRc is set as 1.0. The results represent the means of three independent experiments. Error bars indicate the SD (n = 3). Asterisks indicate significant differences in the translational efficiency of UTRc at P < 0.05.
Fig 5.
Schematic representation of putative cis-acting RNA regulatory elements of UTRc that can impact the expression of OsMac1 mRNA variants.
Regions of the predicted intramolecular interactions of UTRc are shown. SL1, SL2, SL3, and SL4 indicate the predicted stem-loop structures. Region–A and Region–B indicate the predicted intramolecular interactions. sp38 is colored orange. Initiation and termination codons of uORFs are colored pink. Local secondary structures of UTRb and UTRc were predicted using the CentroidFold program (http://www.ncrna.org/centroidfold). The nucleotide sequence of UTRc is shown in S3 Fig.
Fig 6.
Dependence of the OsMac1 mRNA translation efficiency on secondary and tertiary UTRc structures.
(A) Disruption of SL3 stem section 1 is dispensable for UTRc function. Schematic representation of stem section 1 of SL3 (upper panel). Complementary mutations in the 5’ arm of stem section 1 are shown by red letters, UTRc (ΔSL3). (B) Translational efficiency of SL4 mutants. Schematic representation of entire mutated and truncated SL4 (upper panels). "Truncation" indicates a deletion mutant lacking the stem-loop as indicated. (C) Schematic representation of a base-paired motif between Region-B (nt 564–559) and UTRc (nt 242–250) (upper panels). Two sets of mutations are shown: those introduced as complementary to destroy base pairing ("Complementary") and the additional complementary mutations introduced to rebuild the base pairings ("Restored-interaction"). Translational efficiencies are shown as the relative GUS activities that are normalized by the amount of mRNA. The value of the GUS activity of UTRc is set as 1.0. The results represent the means of three independent experiments. Error bars indicate the SD (n = 3). Asterisks indicate significant differences in the translational efficiency of UTRc at P < 0.05.