Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition
- Institut de Biologie Moléculaire et Cellulaire, “Architecture et Réactivité de l’ARN” CNRS UPR9002, Université de Strasbourg, France
Figures
Figure 1 with 1 supplement
Translation inhibitory element (TIE)-mediated inhibition is recapitulated in rabbit reticulocyte lysate (RRL) and does not require full-length TIEs.
(A) Three capped mRNAs were used to test TIE-mediated inhibition in vitro. Hoxa3 TIE and Hoxa11 TIE were placed upstream of the 5’UTR of Hbb-b1 and the Renilla luciferase coding sequence. …
Figure 1—figure supplement 1
Translation inhibitory element (TIE)-mediated inhibition recapitulated in different in vitro and in vivo systems.
(A) Translation efficiency was measured with a 5’UTR Hbb-b1 Renilla luciferase (RLuc) reporter without TIE. A-capped and m7G-capped mRNAs were first used to assess cap dependency of the rabbit …
Figure 2 with 1 supplement
The secondary structural models of Hoxa3 translation inhibitory element (TIE) and Hoxa11 TIE reveal distinct structures.
The structures of (A) Hoxa3 TIE (170 nucleotides) and (B) Hoxa11 TIE (216 nucleotides) were obtained by chemical probing using base-specific reagents, dimethyl sulfate (DMS) and …
Figure 2—figure supplement 1
Average of reactivities of dimethyl sulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) for Hoxa3 translation inhibitory element (TIE) and Hoxa11 TIE.
Probing experiments by DMS and CMCT were performed in triplicates. Figures S2A and S2B represent averages of reactivities of DMS and CMCT for Hoxa3 TIE, respectively. Figures S2C and S2D represent …
Figure 3 with 1 supplement
Upstream AUG111 in Hoxa3 translation inhibitory element (TIE) is essential for inhibition.
(A) Substitution mutations in uAUG111and uAUG123 to UAC in Hoxa3 TIE were performed. Constructs with the corresponding mutations were translated in rabbit reticulocyte lysate (RRL) and luciferase …
Figure 3—figure supplement 1
Upstream AUG111 in Hoxa3 translation inhibitory element (TIE) is essential for inhibition.
(A) (To the left) AUG-like mutations (AUU, GUG, CUG, and ACG) were tested in vitro in rabbit reticulocyte lysate (RRL) compared to wt Hoxa3. (To the right) Representation of uAUG111 context in the …
Figure 4 with 1 supplement
The uAUG111 in Hoxa3 translation inhibitory element (TIE) is translated through 5’UTR of Hoxa3.
(A) Three transcripts were used for this experiment: full-length 5’UTR of Hoxa3, a deletion mutant at nucleotide G333 in Hoxa3 internal ribosome entry site (IRES) and a control transcript without …
Figure 4—figure supplement 1
Translation of Hoxa3 translation inhibitory element (TIE) upstream open reading frame (uORF) in different constructs.
(A) A single nucleotide deletion of A220 in Hoxa3 TIE is performed to frameshift the uORF frame to the same as the main Renilla luciferase (RLuc) ORF. Another mutation was performed combining this …
Figure 5 with 2 supplements
A start-stop upstream open reading frame (uORF) in Hoxa11 translation inhibitory element (TIE) stalls an 80S upstream of a highly stable structure.
(A) Mutational analysis of uAUGs in Hoxa11 TIE. Three transcripts with AUG/UAC mutations were used: M1: (AUG/UAC)84 + (AUG/UAC)159, M2: (AUG/UAC)84, and M3: AUG/UAC159. Transcripts were translated …
Figure 5—figure supplement 1
Transplanting Hoxa11 translation inhibitory element (TIE) stem loop structure in Hbb-b1 5’ UTR efficiently inhibits translation of Renilla luciferase (RLuc) mRNA.
The stem loop structure of Hoxa11 TIE (104–154) was transplanted in the 5’UTR of Hbb-b1 with 25 nucleotides spanning from each extremity. The three shown transcripts were in vitro translated in …
Figure 5—figure supplement 2
Polysome fractionation on 7–47% sucrose gradient of pre-initiation complexes.
Polysome fractionation of pre-initiation complexes programmed with m7G-capped radioactive mRNAs containing Wt Hoxa3 translation inhibitory element (TIE) and the mutant (AUG/UAC)84 in rabbit …
Figure 6 with 1 supplement
Distinct profiles for factors involved in translation inhibitory element (TIE)-mediated inhibition blocked with cycloheximide.
Mass spectrometry analysis of cycloheximide-blocked translation initiation complexes and on three transcripts: Hoxa3 TIE, Hoxa11 TIE placed upstream of 5’UTR of Hbb-b1, and the 5’UTR of Hbb-b1 …
Figure 6—figure supplement 1
Distinct profiles for factors involved in translation inhibitory element (TIE)-mediated inhibition blocked with GMP-PNP.
Mass spectrometry analysis of GMP-PNP-blocked translation initiation complexes and on three transcripts: Hoxa3 TIE, Hoxa11 TIE placed upstream of 5’UTR of Hbb-b1, and the 5’UTR of Hbb-b1 (control). …
Figure 7
Co-transfection assay of translation inhibitory element (TIE) plasmids and siRNA against eIF2D confirms its implication in Hoxa3-mediated inhibition.
Mutational analysis of A-motif sequences in Hoxa3 TIE shows a requirement for an upstream A-motif for efficient inhibition. (A) Co-transfection assay was performed using pmirGLO plasmids with …
Figure 8 with 1 supplement
Two distinct models for translational inhibition by Hoxa3 translation inhibitory element (TIE) and Hoxa11 TIE.
A model for Hoxa3 TIE suggests a ribosomal assembly on the uAUG111 with a requirement of eIF2D initiation factor. The model for Hoxa11 TIE suggests a stalled 80S ribosome on AUG-stop codons …
Figure 8—figure supplement 1
Alignment of Hoxa3 translation inhibitory element (TIE) and Hoxa11 TIE among different species shows variation in the conservation of the inhibitory elements.
(A) Nucleotides 1–170 from the mouse Hoxa3 TIE sequence were aligned with different species. The position of uAUG in Mus musculus is highlighted in a red box. Accession numbers: Felis catus: …
Additional files
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Supplementary file 1
Mass spectrometry analysis of pre-initiation complexes programmed with Hoxa3 TIE, Hoxa11 TIE, and 5’UTR Hbb-b1 in the presence of cycloheximide.
- https://cdn.elifesciences.org/articles/66369/elife-66369-supp1-v1.xls
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Supplementary file 2
Mass spectrometry analysis of pre-initiation complexes programmed with Hoxa3 TIE, Hoxa11 TIE, and 5’UTR Hbb-b1 in the presence of GMP-PNP.
- https://cdn.elifesciences.org/articles/66369/elife-66369-supp2-v1.xls
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66369/elife-66369-transrepform-v1.docx
