Article Figures & Data
Figures
- FIG. 1.
Optimal secondary structure of the 5′-UTR of GLV mRNA predicted by the minimum free-energy minimization program MFOLD (14, 30). The stem-loop structures were designated U1 to U5. The boxed region indicates the initiation codon at the downstream end of 5′-UTR.
- FIG. 2.
Structural probing of stem-loop U3 and the pseudoknot structure with chemical modification and enzymatic digestion. (A) Chemical modification of A and C (by DMS), G (by KE), and U and G (by CMCT) was monitored by reverse transcription with a radiolabeled primer hybridizing to positions 194 to 211 in the 5′-UTR. Durations for chemical modification (in minutes) are indicated above each lane. Bases indicated on the right side of the gel are chemically modified. (B) Products from RNaseV1 (V1) and RNaseT1 (T1) digestion were analyzed in primer extension as described above. The units of RNase used are indicated by the numbers above each lane. Bases identified on the right side of the figure indicate points of digestion by RNaseV1 and/or RNase T1. ♦, an unusual digestion of A residue by RNase T1; *, CMCT-modified A. DNA ladders on the left are for base identification.
- FIG. 3.
The proposed pseudoknot structure. Chemically modified bases are boxed, the sites of strong RNaseV1 digestion are indicated by pentagons, and strong RNaseT1 digestion is indicated by arrows.
- FIG. 4.
Chemical modification and enzymatic digestion of the predicted stem-loop U4a. Chemical modification (A) and enzyme digestion (B) were monitored by reverse transcription with a radiolabeled primer hybridizing to positions 242 to 259 in the 5′-UTR. Bases indicated on the right sides of the figures are modified by chemicals (A) or digested by ribonucleases (B). The symbol ♦ indicates an unusual digestion of U residue by RNase T1. DNA ladders on the left are for base identification.
- FIG. 5.
Chemical modification and enzymatic digestion of the predicted stem-loop U4b. Chemical modification (A) and enzyme digestion (B) were monitored by reverse transcription with a radiolabeled primer hybridizing to positions 304 to 322 in the 5′-UTR. Bases indicated on the right sides of the figures are modified by chemicals (A) or digested by enzymes (B). DNA ladders on the left are for base identification.
- FIG. 6.
Chemical modification and enzymatic digestion of the predicted stem-loop U4c. Chemical modification (A) and enzyme digestion (B) were monitored by reverse transcription with a radiolabeled primer hybridizing to positions 304 to 322 in the 5′-UTR. Bases on the right sides of the figures are chemically modified (A) or enzymatically digested (B). DNA ladders on the left are for base identification.
- FIG. 7.
Chemical and enzymatic probing of the predicted stem-loop U5. Chemical modification (A) and enzyme digestion (B) were monitored by reverse transcription with a radiolabeled primer hybridizing to positions 369 to 386 in the capsid coding region. Bases on the right sides of the figures are chemically modified (A) or enzymatically digested (B). The symbol (♦) indicates an unusual digestion of U residue by RNase T1. The ladders on the left are for base identification. The sensitivity of some of the residues (C315 to C318, G323 to C324, and C332) to chemical modification cannot be resolved due the presence of background in the control lanes (*). They have been attributed to strong structural features that cause the premature termination of reverse transcription (20).
- FIG. 8.
The secondary structure of GLV mRNA 5′-UTR derived from the experimental results shown in Fig. 2 to 7. Numbers indicate nucleotide positions in the 5′-UTR. Chemically modified bases are boxed, sites of RNaseV1 digestion are indicated by pentagons, and sites of strong RNaseT1 digestion are indicated by arrows.
- FIG. 9.
Structures of individual MFOLD-predicted stem-loops U3, U4a, U4b, U4c, and U5 as indicated in Fig. 1, with arrows indicating the positions of individual site-directed mutations. Results from analyzing these mutants are presented in Table 1.
- FIG. 10.
Northern blot analysis showing varied stabilities among the stem-loop U3 (A) and pseudoknot mutant (B) transcripts in Giardia cells. Total RNA was extracted from transfected Giardia trophozoites 16 h after electroporation and analyzed by Northern blotting using [α-32P]-luc DNA as a probe. The same samples, stained with ethidium bromide in gel, were used as sampling controls. Mutations are indicated on the top of each lane for stem-loop U3 mutants (A) and pseudoknot mutants (B) (see Table 2). The transcript level determined for each mutant (see Materials and Methods) is indicated on the bottom of each lane.
- FIG. 11.
Secondary structure of GLV IRES verified by experimental data.
Tables
- TABLE 1.
Relative luciferase activities of mutant transcript-transfected Giardia and the stability of the mutant transcripts
Stem-loop and mutation RLU (%)a Remark Stabilityc Stem-loop U3 100 + C139G/C140G 2.1 ± 0.2 Wobbled stem − G158C/G159C 9.2 ± 4.2 Wobbled stem − C139G/C140G-G158C/G159Cb 139.0 ± 17.5 Restored stem + Δ 134-164 1.0 ± 0.2 Entire U3 deleted − Δ 134-175 1.2 ± 0.14 Entire pseudoknot deleted − Stem-loop U4a 100 + G205C/G206C 17.3 ± 0.9 Wobbled stem + G205C/G206C-C217G/C218Gb 4.8 ± 0.2 Restored stem + C217A 94.6 ± 5.4 A bulge in stem ND G206C/G207C 2.7 ± 0.6 Disrupted stem ND G208C/G209C 1.0 ± 0.05 Loop mutation + A210C/A211C 17.2 ± 0.6 Loop mutation ND U212C/U213C 54.7 ± 1.5 Loop mutation ND Δ 204-219 7.7 ± 0.8 Entire U4a deleted + Stem-loop U4b 100 + U225C 58.3 ± 6.0 Shortened stem ND U237A/G238C 63.5 ± 4.0 Loop mutation ND G240C/G241C 54.4 ± 5.7 Loop mutation ND C248G/C249G 7.4 ± 0.2 Loop mutation + C250U/G251A 10.6 ± 0.5 Loop mutation ND C244U/A245G 11.7 ± 2.0 Loop mutation + Δ 221-261 12.6 ± 0.8 Entire U4b deleted + Stem-loop U4c 100 + G271C/G272C/G273C 88.6 ± 6.6 Wobbled stem ND Δ 263-292 30.5 ± 2.1 Entire U4c deleted + Stem-loop U5 100 + C316G/C317G 0.04 ± 0.01 Wobbled stem + G341C/G342C 0.3 ± 0.1 Wobbled stem + C316G/C317G-G341C/G342Cb 92.6 ± 3.4 Restored stem + Δ 314-344 0.1 ± 0.02 Entire U5 deleted + ↵ a Values are averages of relative luciferase activities obtained from at least two independent transfection experiments with the wild type (pC631luc) as the positive control (100%). The wild type and the mutants were all transfected in triplicate in each experiment.
↵ b Restorative mutations.
↵ c Stability of the transcripts as determined by Northern blot analysis. −, loss of the transcript; +, presence of the transcript. See Fig. 10 for stem-loop U3 and Fig. 1 in the supplemental material for stem-loops U4a to U5. ND, not determined.
- TABLE 2.
Relative luciferase activities expressed by pseudoknot mutant transcript-transfected Giardiaa
Mutation nt 147-154 nt 168-175 RLU (%)b Stabilityc None (wild type) AACACAUA UAUGUGUU 100 + 1A cACACAUA UAUGUGUU 23.9 ± 2.8 − 1B AACACAUA UAUGUGUg 23.5 ± 1.7 − 1R cACACAUA UAUGUGUg 86.5 ± 8.2 + 2A AcaACAUA UAUGUGUU 2.1 ± 0.3 − 2B AACACAUA UAUGUugU 12.4 ± 0.8 − 2R AcaACAUA UAUGUugU 125.1 ± 1.6 + 3A AAgugAUA UAUGUGUU 2.5 ± 1.6 − 3B AACACAUA UAUcacUU 2.5 ± 0.1 − 3R AAgugAUA UAUcacUU 54.3 ± 2.1 + 4A AACAugUA UAUGUGUU 1.9 ± 0.2 − 4B AACACAUA UAcaUGUU 2.7 ± 0.1 − 4R AACAugUA UAcaUGUU 49.7 ± 3.7 − 5A AACACAcA UAUGUGUU 5.5 ± 0.7 − 5B AACACAUA UgUGUGUU 9.7 ± 0.5 − 5R AACACAcA UgUGUGUU 3.1 ± 0.3 − 6A AACACAUc UAUGUGUU 1.3 ± 0.1 − 6B AACACAUA gAUGUGUU 0.2 ± 0.1 − 6R AACACAUc gAUGUGUU 0.6 ± 0.1 − ↵ a The letters in boldface lowercase are the substituted bases.
↵ b Each value is an average from at least two independent transfection experiments with the wild type (pC631luc) as positive control (100%). The wild type and the mutants were all used to transfect cells in triplicate in each experiment.
↵ c Stability of the transcripts as determined by Northern blot analysis. −, loss of the transcript; +, presence of the transcript. See Fig. 10 for details.
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Files in this Data Supplement:
- Supplemental file 1
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Supplementary FIG. 1. Northern blot analysis showing unchanged stability among mutant U4a, U4b, U4c, and U5 transcripts
in Giardia cells. Total RNA was extracted from transfected Giardia trophozoites 16 hours after electroporation and analyzed in northern blot using [α-32P]-luc DNA as a probe. The same samples stained with ethidium bromide in gel were used as sampling controls.
PowerPoint presentation, 102K.
- Supplemental file 1
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Supplementary FIG. 1. Northern blot analysis showing unchanged stability among mutant U4a, U4b, U4c, and U5 transcripts
in Giardia cells. Total RNA was extracted from transfected Giardia trophozoites 16 hours after electroporation and analyzed in northern blot using [α-32P]-luc DNA as a probe. The same samples stained with ethidium bromide in gel were used as sampling controls.
