1. Biochemistry and Chemical Biology
  2. Cell Biology
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Puromycin reactivity does not accurately localize translation at the subcellular level

  1. Syed Usman Enam
  2. Boris Zinshteyn
  3. Daniel H Goldman
  4. Madeline Cassani
  5. Nathan M Livingston
  6. Geraldine Seydoux
  7. Rachel Green  Is a corresponding author
  1. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, United States
  2. Howard Hughes Medical Institute, United States
  3. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, United States
Research Article
Cite this article as: eLife 2020;9:e60303 doi: 10.7554/eLife.60303
5 figures, 1 table and 3 additional files

Figures

Mechanisms of action of puromycin and other translational inhibitors.

(A) Comparison of structure of 3′ terminus of tyrosyl tRNA with that of puromycin. Key differences are highlighted in green. tRNA body not drawn to scale. (B) Scheme for reaction of puromycin with peptidyl P-site tRNA on the ribosome, leading to dissociation of puromycylated peptide. (C) Structures and schematicized ribosome binding sites of translational inhibitors cycloheximide, anisomycin and emetine. Binding sites are based on Garreau de Loubresse et al., 2014; Wong et al., 2014.

O-propargyl-puromycin (OPP) labels nuclei in the distal germline of C. elegans in the presence or absence of emetine.

(A) Representative photomicrographs of germlines labeled for 5 min with 20 µM OPP, and pre-treated for 15 min with control buffer (top row), 45 µM emetine (second row), or 37 µM anisomycin (bottom row). DAPI labels chromosomes. Post-fixation, click labeling of OPP with Alexa Fluor 488 picolyl azide revealed OPP throughout the cytoplasm and concentrated in nuclei. Scale bar = 10 µm. (B) Quantification of OPP-Alexa 488 signal in distal germlines. Each dot represents the average fluorescence of the mitotic zone of one worm germline. Values are normalized to the average obtained for germlines pre-treated with control buffer (OPP alone). P values were obtained through an unpaired t-test. Experiment performed in duplicate.

Figure 3 with 1 supplement
Emetine does not prevent release of puromycylated luciferase from rabbit reticulocyte ribosomes.

(A) Schematic of the real-time translation monitoring assay in rabbit reticulocyte lysate. (1) (Purple trace) Ribosomes translate the full-length luciferase mRNA and release luciferase which becomes enzymatically active and results in an increase in luminescence. (Yellow trace) Ribosomes stall at the 3’ end of a truncated luciferase mRNA and little to no luminescence is observed as the ribosome-bound luciferase peptides are in an enzymatically inactive conformation. (2) Puromycin (PM) is added to the system, stopping further translation and causing all nascent peptides to release from the ribosomes. (3) (Yellow) The luciferase rapidly folds into an enzymatically active conformation and a substantial increase in luminescence is observed. (B) Either puromycin (yellow), H2O (purple) or a mixture of emetine (EME) and puromycin (blue) was added to a reaction containing truncated luciferase mRNA at t = 21 min. Experiment was performed in duplicate; mean traces shown as solid lines and range of replicates shaded. (C) GDPNP was added to a reaction containing truncated luciferase mRNA at t = 16 min for 5 min to inhibit translation across samples. Then, either emetine (blue, purple), anisomycin (ANS) (green) or H2O (yellow) was added to the reaction followed by puromycin (blue, yellow, green) or H2O (purple) 5 min later. Experiment was performed in duplicate; mean traces shown as solid lines and range of replicates shaded. Note that the experiments in (A and B), and Figure 3—figure supplement 1B were done in the same batch, and the yellow traces (PM treated) in these panels are the same.

Figure 3—figure supplement 1
Additional control experiments for lysate-based luciferase assays.

(A) Luminescence measured from translation of full-length luciferase mRNA in rabbit reticulocyte lysate. Either emetine (blue), anisomycin (green) or puromycin (yellow) was added to the reaction at indicated concentrations. Due to experimental restrictions, emetine and anisomycin were added at t = 16 min and puromycin was added at t = 22 min. Experiment performed once. (B) Luminescence measured from translation of truncated luciferase mRNA in rabbit reticulocyte lysate. Either emetine (blue), puromycin (yellow) or anisomycin (green) was added to the reaction at t = 21 min for 5 min to inhibit translation across the samples. Then, either puromycin (blue, green) or H2O (yellow) was added to the reaction. Experiment was performed in duplicate; mean traces shown as solid lines and range of replicates shaded.

Puromycin treatment causes loss of nascent peptide-mRNA co-localization, independent of elongation inhibitors.

(A) SunTag reporter schematic. In addition to the tandem SunTag repeats and the auxin-inducible degron, this reporter encodes nano luciferase and BFP, which are not used in the present experiments. The 3’ UTR also encodes tandem repeats of the MS2 stem loop, which can be used to label the mRNA red. However, since we detect mRNA by FISH, we do not use the MS2 stem loops in the present experiments. (B) Example cells imaged by FISH-IF. Cells were either untreated (top row), treated with 91 μM puromycin for 5 min (second row), pre-treated with 208 μM emetine for 15 min followed by 91 μM puromycin for 5 min (third row), or pre-treated with 37 μM anisomycin for 5 min followed by 91 μM puromycin for 5 min (last row). Yellow arrows: examples of translating mRNAs; White arrows: example of single fully synthesized SunTag polypeptide (released from the ribosome); Blue arrows: examples of untranslating mRNAs. Scale bar in top left image: 10 microns. Scale bar in top right image: two microns. (C) Fraction of mRNAs co-localized with SunTag signal. Each dot represents one cell. Cells are only included in the analysis if they have more than five and fewer than 36 mRNAs. 20–27 cells and 313–513 mRNAs per condition were analyzed. Black lines indicate mean with standard error of the mean. P values were calculated by two-sample t-test. Experiment performed once.

Proteins diffuse over long distances in the cell during common puromycin labeling times.

Calculation of expected displacement by diffusion as a function of time, using the equation < x2 > = 2nDt where n is the dimensionality, D is the diffusion coefficient (126 µm2/s Di Rienzo et al., 2014) and t is time. The calculation is shown for 1, 2 and 3 dimensions.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Recombinant DNA reagentpGEM-luc (plasmid)PromegaGenBank X65316.2Firefly luciferase cassette vector
Recombinant DNA reagentpSL312 (plasmid)This paperFull-length firefly luciferase template; can be obtained from Green Lab
Recombinant DNA reagentP3.35_pGEM_luc_trunc_kozak_RC
(plasmid)
This paperTruncated firefly luciferase template; can be obtained from Green Lab
Peptide, recombinant proteinStuI
(restriction enzyme)
NEBR0187SLinearization of pSL312 for SP6 transcription
Peptide, recombinant proteinHpaI
(restriction enzyme)
NEBR0105SLinearization of P3.35 for SP6 transcription
Sequence-based reagentFull-length luciferase mRNAThis paperSP6 transcribed from pSL312
Sequence-based reagentTruncated luciferase mRNAThis paperSP6 transcribed from P3.35
Commercial assay or kitmMESSAGE mMACHINE SP6 transcription kitInvitrogenAM1340
Commercial assay or kitNuclease-treatedrabbit reticulocyte lysate translation reactionsPromegaL4960
Chemical compound, drugLuciferinPerkinElmer122799
Peptide, recombinant proteinSuperase-In RNase InhibitorInvitrogenAM2696
Chemical compound, drug5'-guanylyl imidodiphosphate (GDPNP)Jena BioscienceNU-401–50
Chemical compound, drugEmetineCayman Chemical21048
Chemical compound, drugPuromycinSigma AldrichP7255
Chemical compound, drugAnisomycinSigmaA9789
Genetic reagent
(C. elegans)
N2Caenorhabditis Genetics Center (CGC)
Commercial assay or kitClick-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay kitInvitrogenC10456
Cell line (human)U-2OS cells containing Flp-In locusAndrew Holland lab (Johns Hopkins University)
Chemical compound, drugamino-11–12 ddUTPLumiprobeA5040
Peptide, recombinant proteindeoxynucleotidyl transferaseThermo FisherEP0162
Chemical compound, drugdoxycycline hyclateMillipore SigmaD9891
Chemical compound, drugCy3-NHS esterLumiprobe41020
Chemical compound, drug3-indole acetic acidSigma AldrichI2886
Chemical compound, drugparaformaldehydeElectron Microscopy Sciences50-980-492
Peptide, recombinant proteinrat tail collagen IGibcoA1048301
Peptide, recombinant proteinBSAVWRVWRV0332-25G
Chemical compound, drugSSC bufferCorning46–020 CM
Chemical compound, drugformamideSigma AldrichF9037-100ML
Sequence-based reagentE. coli tRNASigma Aldrich10109541001
Chemical compound, drugdextran sulfateSigma AldrichD8906-100G
Chemical compound, drugribonucleoside vanadyl complexNEBS1402S
AntibodyChicken polyclonal anti-GFP antibodyAves LabsRRID:AB_23073131:1000 dilution
AntibodyGoat anti-chicken polyclonal IgY secondary antibodyThermo FisherRRID:AB_25340961:1000 dilution
Chemical compound, drugProLong Diamond antifade reagentInvitrogenP36962
Recombinant DNA reagentpubc-OSTIR1-IRES-scFv-sfGFP-NLS (plasmid)Reference 32
Software, algorithmFISH-quantReference 45
Software, algorithmCustom MATLAB scripts for processing FISH-quant outputThis paperScripts for quantifying number of translating ribosomes per mRNA from FISH-IF data; available asSource code 1 in supplementary files
Sequence-based reagentOligonucleotides used to generate FISH probesReference 32See Supplementary file 1

Data availability

Raw data for all plots have been deposited in accompanying excel files.

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