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, 36 (20), 2968-2986

Dual Function of UPF3B in Early and Late Translation Termination

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Dual Function of UPF3B in Early and Late Translation Termination

Gabriele Neu-Yilik et al. EMBO J.

Abstract

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD-related diseases. Here, we have used a fully reconstituted in vitro translation system to probe the NMD proteins for interaction with the termination apparatus. We discovered that UPF3B (i) interacts with the release factors, (ii) delays translation termination and (iii) dissociates post-termination ribosomal complexes that are devoid of the nascent peptide. Furthermore, we identified UPF1 and ribosomes as new interaction partners of UPF3B. These previously unknown functions of UPF3B during the early and late phases of translation termination suggest that UPF3B is involved in the crosstalk between the NMD machinery and the PTC-bound ribosome, a central mechanistic step of RNA surveillance.

Keywords: UPF3B; nonsense‐mediated mRNA decay; translation termination.

Figures

Figure EV1
Figure EV1. Validation of the experimental system

SDS–PAGE analysis of purified recombinant proteins used in the toeprinting experiments as indicated.

Toeprint analysis of translation complexes prepared by the reconstituted in vitro translation system. Above: 80S initiation complex; middle: pre‐termination complex (pre‐TC); below: termination complex formed in presence of eRF1, eRF3a and GTP. Peaks at 138 nt indicate the position of the 80S initiation complex on mRNA, peaks at 129 nt indicate the position of pre‐TC and peaks at 127 nt correspond to the termination complex (post‐TC). Rfu—relative fluorescence units.

Schematic representation of UPF1 variants used in (D).

Thin layer chromatography (TLC) analysis of the ATPase activity of UPF1 variants in the absence or presence of UPF2L and/or UPF3B at 30°C in MES buffer (pH 6.5, lanes 1–7) or translation buffer (pH 7.5, lanes 8–13), respectively. 1.5 μl of the samples was spotted on the TLC plates, and the residual 18.5 μl was analysed on SDS–PAGE gels for loading control (lower panels). The positions of γ32P‐ATP and γ32Pi are indicated. % ATP hydrolysis in (D and E) was calculated using a phosphoimager and displays the means ± SEM of four independent experiments.

ATP hydrolysis experiment as in (D) at 37°C in translation buffer.

Source data are available online for this figure.
Figure 1
Figure 1. UPF3B delays translation termination in vitro

Scheme of the MVHC‐STOP mRNA.

Toeprinting analysis of ribosomal complexes obtained by incubating pre‐TCs assembled on MVHC‐STOP mRNA (MVHC‐pre‐TCs) with UPF1, UPF2L, UPF3B or BSA at 1 mM free Mg2+ followed by termination with limiting amounts of eRF1 and eRF3a. The positions of pre‐TCs, post‐TCs and full‐length cDNA are indicated. In all toeprinting analyses the box marked TAA indicates the stop codon in the cDNA sequence corresponding to the MVHC‐STOP mRNA. Asterisks mark initiation and elongation complexes in all toeprinting analyses. Representative of five independent experiments. Lanes C, T, A, G indicates the sequence of the cDNA derived from the MVHC‐STOP mRNA using the same primer as the toeprint experiment. The box ‘TAA’ indicates the position of the stop codon.

Kinetics of [35S]‐peptidyl‐tRNA hydrolysis in the presence of eRF1 and eRF3a (black circles) or eRF1, eRF3a and UPF3B (white triangles). Termination reactions were assembled as in (B). A value equal to 1 corresponds to the maximum value for peptide release triggered by eRF1 and eRF3a. Data points show the mean of three experiments ± SEM.

UPF1 in vitro phosphorylation by SMG1‐8‐9 in the presence of UPF2L and/or UPF3B and in the presence (lanes 7–12) or absence (lanes 1–6) of the eRFs. In lanes 13–16 UPF2L and/or UPF3B were added after UPF1 phosphorylation. Samples were analysed by SDS–PAGE, Coomassie‐stained to control for equal loading (lower panel) and autoradiographed (upper panel). SMG1 autophosphorylation (P‐SMG1) confirms equal SMG1‐activity in all samples. UPF1 is represented by the lower and UPF2L by the upper of the two closely migrating bands between 125 and 130 kDa in the Coomassie‐stained gel. Representative of two independent experiments.

Toeprinting analysis of ribosomal complexes obtained by incubating pre‐TCs with UPF1, UPF2L, UPF3B, SMG1‐8‐9 or BSA at 1 mM free Mg2+ as indicated followed by translation termination by eRF1 and eRF3a. See also Fig EV2. Representative of three independent experiments.

Source data are available online for this figure.
Figure EV2
Figure EV2. Validation of the termination‐delaying effect of UPF3B

Toeprinting analysis of ribosomal complexes obtained by incubating MVHC‐pre‐TCs at 1 mM free Mg2+ with decreasing amounts of eRFs. Representative example for the titration of eRF1 and eRF3a to identify concentrations slowing down the pre‐TC to post‐TC transition. The amount used for the sample in lane 5 was chosen for further experiments with this batch of pre‐TCs.

Toeprinting analysis of ribosomal complexes obtained by incubating MVHC‐pre‐TCs with UPF3B, eIF4B, IRP1, SXL or BSA at 1 mM free Mg2+ and 1 mM ATP followed by termination with limiting amounts of eRF1 and eRF3a.

Toeprinting analysis of ribosomal complexes obtained as in (B) by incubating MVHC‐pre‐TCs with UPF3B, UPF3B‐N, UPF3B‐M, UPF3BΔEBD or BSA.

Toeprinting analysis of ribosomal complexes obtained as in Fig 1B in the presence of 1 mM ATP or AMPPNP, respectively.

Data information: Asterisks mark initiation and elongation complexes. Panels (A, B) each represent two independent experiments. Panel (D) represents three independent experiments. Source data are available online for this figure.
Figure 2
Figure 2. In vivo interaction between release factors and UPF proteins

Co‐immunoprecipitation from RNaseA‐treated lysates of HeLa cells transfected with FLAG‐eRF1 (lanes 1–10) or unfused FLAG (lanes 11–15) and V5‐eRF3a, V5‐UPF1, V5‐UPF2, V5‐UPF3B or V5‐PYM. Co‐precipitated proteins were detected using an anti‐V5 antibody. Lysate used for the immunoprecipitations was loaded in the input lanes (left). Re‐probing with anti‐TUBB antibody served as loading control.

Co‐IP experiment as in (A) with FLAG‐eRF3a. Re‐probing with anti‐ACTB served as loading control.

Data information: Each panel represent two independent experiments. Because TUBB migrates at virtually the same position as FLAG‐eRF3a and ACTB migrates very closely to FLAG‐eRF1, TUBB was used as loading control for (A) and ACTB for (B). Source data are available online for this figure.
Figure 3
Figure 3. UPF3B forms a complex with eRF3a and eRF1

Schematic representation of eRFs, UPF1, UPF2L and UPF3B proteins. Domains of known function and structural motifs are indicated. N, M, C, G, CH stand for N‐terminal, middle, C‐terminal, GTP‐binding and cysteine–histidine‐rich domain, respectively. 2/3: domains 2 and 3. MIF4G: middle fragment of eIF4G, RRM: RNA recognition motif, EBM: EJC‐binding motif.

In vitro pulldown of eRF1 and/or eRF3a with His‐UPF1. Protein mixtures before loading onto the beads (input) or after elution (eluate) were separated by SDS–PAGE.

Pulldown as in (B), with His‐UPF2L as bait.

Pulldown as in (B), with His‐UPF3B as bait.

Pulldown of eRF1, UPF3B, or both with His‐eRF3a in buffer containing 0 (lane 4), 2.5 (lane 5) or 5 mM (lane 6) Mg2+.

Left: SEC elution profile of eRF3a (yellow), UPF3B (green), or both (blue). The elution volume (in ml) is indicated for each experiment. Column calibration was performed with globular proteins (shown above). Right: SDS–PAGE analysis of eluate fractions. M: protein molecular weight standards (kDa).

SEC elution profile and SDS–PAGE analysis as in (F) of eRF1 (red), eRF3a (orange), UPF3B (dark green) or all three (light green). See also Fig EV3.

Data information: Panels (B, C) each represent three independent experiments. Panels (E–G) each represent two independent experiments. (B–D) Bands in lanes 1, 2 and 4 of the eluate panels represent background binding of the untagged eRFs to the Ni‐NTA resin. Source data are available online for this figure.
Figure EV3
Figure EV3. Validation of UPF3B's complex formation with eRF1 and eRF3a

In vitro pulldown of eRF1 and/or UPF1 with His‐eRF3a. Protein mixtures before loading onto the beads (input) or after elution (eluate) were separated by SDS–PAGE.

Pull‐down experiment as in (A) with eRF1, UPF3B and His‐eRF3a.

Pull‐down experiment as in (B) with eRF1, UPF1 and His‐eRF3a in buffer containing 0, 2.5 or 5 mM Mg2+ (lanes 4, 5, or 6 respectively).

Molecular mass of UPF3B determined by size‐exclusion chromatography using a Superdex 200 column combined with detection by multiangle laser light scattering and refractometry (SEC‐MALLS‐RI). The SEC elution profiles as monitored by refractometry (RI) are represented for UPF3B. The molecular mass (MM) of UPF3B calculated from light scattering and refractometry data is indicated.

Left: SEC elution profile of eRF1 (red), UPF3B (green) or both (blue). The elution volume (in ml) is indicated for each experiment. Calibration of the column was performed with globular proteins (shown above). Right: SDS–PAGE analysis of eluate fractions. M: protein molecular weight standards (kDa).

Data information: Panels (A–C) each represent three independent experiments. Panels (D, E) each represent two independent experiments. (A–C) Bands in lanes 1, 3 and 5 of the eluate panels (A, B) and bands in lanes 1 and 3 of the eluate panel in (C) represent background binding of untagged proteins to the Ni‐NTA resin. Source data are available online for this figure.
Figure 4
Figure 4. UPF3B can directly interact with UPF1

In vitro pulldown of eRF3a, UPF1 or both with His‐UPF3B. Protein mixtures before loading onto the beads (input) or after elution (eluate) were separated by SDS–PAGE. Representative of four independent experiments.

Left: SEC elution profile of UPF1 (purple), UPF3B (green) or both (orange). Right: SDS–PAGE analysis of eluate fractions. Representative of two independent experiments. Since the experiments described in Figs 3F and 4B were performed in parallel, the same UPF3B SEC elution profile (green) and the corresponding SDS–PAGE analysis served as control for both experiments. More UPF3B SEC experiments are depicted in Figs 3G, 6B and EV3E.

Source data are available online for this figure.
Figure EV4
Figure EV4. UPF1‐UPF3B complex formation is not prevented by RNA

SEC elution profile of UPF3B (green), UPF1 (purple), RNA (red) and a mix of UPF3B, UPF1 and a threefold excess of RNA (blue). Below: SDS–PAGE analysis of eluate fractions.

Analysis of SEC peak fractions. Peaks representing UPF3B (green) and UPF1 (purple), elute with an OD 260 nm/280 nm ratio of 0.54 and 0.51, respectively, whereas the RNA oligomer (red) elutes with an OD 260 nm/280 nm ratio of 2.0. The peak containing the UPF3B‐UPF1 complex after incubation of UPF1, UPF3B and RNA (blue) has a higher OD 260 nm/280 nm ratio of 0.76 due to the presence of RNA in this peak.

Source data are available online for this figure.
Figure 5
Figure 5. UPF3B interacts with the N‐terminus of eRF3a

Schematic representation of eRF3a constructs used for this Figure. eRF3aΔN, eRF3aΔN199: eRF3a variants lacking amino acids 1–100 and 1–199, respectively.

In vitro pulldown as in Fig 3 of eRF3a (FL) or eRF3aΔN (ΔN) with His‐UPF3B.

Pulldown of UPF3B with His‐eRF3a (FL) or His‐eRF3aΔN.

Schematic representation of UPF3B constructs used in panel (E).

Pulldown as in Fig 3 of eRF3a with His‐UPF3B variants. Asterisks mark the presence of a degradation product in the UPF3B‐N sample.

Co‐IP experiment as in Fig 2 with FLAG‐eRF3aΔ199 and V5‐UPF1, V5‐UPF2L, V5‐UPF3B or V5‐PYM.

Toeprinting analysis of ribosomal complexes obtained by incubating MVHC‐pre‐TCs with BSA or with UPF3B at 1 mM free Mg2+ as indicated. Termination was completed with limiting amounts of eRF1 and either eRF3a (lanes 3, 5) or eRF3aΔN (lanes 4, 6), respectively. Asterisks mark initiation and elongation complexes.

Data information: Panels (B, E) each represent three independent experiments. Panels (C, F, G) each represent two independent experiments. Source data are available online for this figure.
Figure 6
Figure 6. UPF3B binds RNA and ribosomes

Left panel: SEC elution profile of UPF3B (dotted lines), RNA (dashed lines) and the mix (solid lines). Optical density was recorded at 280 nm (blue) and at 260 nm (red). 40 μM of UPF3B or RNA oligonucleotide (24mer) or both was loaded onto a Superdex 200 column. The elution volumes are indicated next to the curves. Right panel: SDS–PAGE analysis of eluate fractions.

Sucrose cushion co‐sedimentation analysis of either ribosomes (lanes 1, 2) or of UPF1, UPF2L or UPF3B or of combinations as indicated. After ultracentrifugation, the supernatant (S) and pellet (P) fractions were analysed by SDS–PAGE.

Sucrose cushion co‐sedimentation analysis of UPF1, UPF2L or UPF3B as in (B) but in the presence of 80S ribosomes.

Data information: Panel (A) represents two independent experiments. Panels (B and C) each represent four independent experiments. Source data are available online for this figure.
Figure 7
Figure 7. UPF3B dissociates post‐TCs

Toeprinting analysis of ribosomal complexes obtained by incubating MVHC‐pre‐TCs with UPF1, UPF2L, UPF3B, or BSA at 1 mM free Mg2+ and 1 mM ATP followed by termination with saturating amounts of eRF1 and eRF3a. Asterisks mark initiation and elongation complexes.

Toeprinting analysis of ribosomal complexes obtained by incubating pre‐TCs as in (A) with UPF3B or BSA and combinations of eRF1, eRF1AGQ, eRF3a and puromycin in the presence of GTP or GMPPNP. Pre‐/post‐TC profiles of lanes 6–8 and 11–14 are enlarged to allow a better assessment. The gel on the left was exposed 2× longer than gel on the right. Note that puromycin‐treated pre‐TCs are relatively unstable at the low Mg2+ concentrations used (Skabkin et al, 2013).

Mg2+ sensitivity of post‐TC dissociation by UPF3B. Toeprinting analysis of ribosomal complexes obtained by incubating MVHC‐pre‐TCs with BSA (lanes 1–6) or UPF3B (lanes 7–12) and at the indicated concentrations of free Mg2+. Termination was completed by adding eRF1 and eRF3a to the samples in lanes 4–9. Lanes 1–6 were exposed 2× longer than lanes 7–12. Asterisks mark initiation and elongation complexes.

Data information: Panel (A) represents three independent experiments. Panels (B, C) each represent two independent experiments. Source data are available online for this figure.
Figure EV5
Figure EV5. UPF3B's post‐TC‐dissolving activity is independent of ATP and SMG1‐8‐9 and requires both the RRM and the middle domain

Toeprinting analysis of ribosomal complexes as in Fig 6A, but in the presence of 1 mM AMPPNP (A) or without adenosine nucleotide (B).

Impact of UPF1‐phosphorylation on efficient translation termination and on ribosome dissociation by UPF3B. Toeprinting analysis of ribosomal complexes obtained by incubating pre‐TCs formed on MVHC‐STOP mRNA (MVHC‐pre‐TCs) with UPF1, UPF2L, UPF3B or BSA at 1 mM free Mg2+ and 1 mM ATP. In lanes 7–10, UPF1 was incubated with SMG1‐8‐9 and ATP for 30 min at 37°C either alone (lane 7) or in the presence of UPF2L, UPF3B, or both (lanes 8–10) before pre‐TCs were added to the mixture and again incubated for 10 min. In lanes 11–14, UPF1 was incubated with ATP and SMG1‐8‐9 for 30 min. Then, UPF2L and/or UPF3B were added for additional 15 min (lanes 12–14). Finally, MVHC‐pre‐TCs were added to the mixtures for 10 min followed by translation termination by eRF1 and eRF3a.

Toeprinting analysis of ribosomal complexes obtained by incubating MVHC‐pre‐TCs with UPF3B, UPF3B‐N, UPF3B‐M, UPF3BΔEBD or BSA at 1 mM free Mg2+ followed by termination with saturating amounts of eRF1 and eRF3a.

Data information: Asterisks mark initiation and elongation complexes. Panel (A) represents three independent experiments. Panels (B, C) each represent two independent experiments.Source data are available online for this figure.
Figure 8
Figure 8. Model for early and late UPF3B function in translation termination
During termination at a PTC ribosome‐bound UPF3B interacts with the eRF1/eRF3a‐GTP complex impeding efficient stop codon recognition. UPF1 bound to the 3′UTR can contact the termination complex, but does not interfere with termination. After GTP hydrolysis and peptide release UPF3B destabilizes the post‐termination ribosomal complex leading to its dissociation. Subsequently, UPF3B, UPF1, UPF2 and other factors activate UPF1's ATPase and helicase functions to remodel the 3′UTR mRNP and attract decay enzymes.

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