Research ArticleMOLECULAR BIOLOGY

Translational control by lysine-encoding A-rich sequences

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Science Advances  24 Jul 2015:
Vol. 1, no. 6, e1500154
DOI: 10.1126/sciadv.1500154
  • Fig. 1 Effects of different lysine codons on mCherry reporter expression and mRNA stability.

    (A) Cartoon of reporter constructs used in electroporation experiments. (B) Western blot analyses of HA-X-mCherry constructs 48 hours after electroporation (HA and β-actin antibodies). (C) Normalized protein expression using LI-COR Western blot analyses or in vivo mCherry fluorescence measurement. β-Actin or fluorescence of coexpressed GFP construct was used for normalization of the data. Each bar represents the percentage of wild-type mCherry (WT) expression/fluorescence. (D) Normalized RNA levels of HA-X-mCherry constructs. Neomycin resistance gene was used for normalization of qRT-PCR data. Each bar represents the percentage of wild-type mCherry (WT) mRNA levels.

  • Fig. 2 The effect of codon usage in polylysine tracks on translation and protein levels.

    (A) Occupancy of ribosomal footprints for regions around different codon combinations for four lysine tracks. All combinations of one, two, three, and four AAG codons per group are shown. Data for four AAA codons are not shown because only a single gene has such a sequence. The upper and lower “hinges” correspond to the first and third quartiles (the 25th and 75th percentiles). The upper and lower whiskers extend from hinges up or down at a maximum of 1.5*IQR (interquartile range) of the respective hinge. (B) Sequences of HA-(A9–A13)-mCherry constructs used in electroporation experiments. (C) Western blot analyses of HA-(A9–A13)-mCherry constructs 48 hours after electroporation (HA and β-actin antibodies). (D) Normalized protein expression using LI-COR Western blot analyses or in vivo mCherry fluorescence measurement. β-Actin or fluorescence of coexpressed GFP construct was used for normalization of the data. Each bar represents the percentage of wild-type mCherry (WT) expression/fluorescence. (E) Normalized RNA levels of HA-X-mCherry constructs. Neomycin resistance gene was used for normalization of qRT-PCR data. Each bar represents the percentage of wild-type mCherry (WT) mRNA levels.

  • Fig. 3 Native poly(A) tracks control reporter mRNA and protein levels.

    (A) Sequences of polylysine runs from human genes incorporated into HA-X-mCherry constructs. Continuous runs of lysine residues are labeled. The number of lysine residues and the ratio of AAG and AAA codons for each construct are indicated. (B) Normalized protein expression using in vivo mCherry reporter fluorescence. Fluorescence of cotransfected GFP was used to normalize the data. Each bar represents the percentage of wild-type mCherry (WT) expression/fluorescence. (C) Normalized RNA levels of HA-X-mCherry constructs. Neomycin resistance gene was used for normalization of qRT-PCR data. Each bar represents the percentage of wild-type mCherry (WT) mRNA levels. (D) Smoothed Gaussian kernel density estimate of positions of poly(A) tracks along the gene. Position of poly(A) segment is expressed as a ratio between the number of the first residue of the poly(A) track and the length of the gene.

  • Fig. 4 The effect of synonymous mutations in poly(A) tracks of human genes.

    (A) Scheme of constructs with ZCRB1 gene poly(A) tracks used for analyses of synonymous mutations. (B) Western blot analyses and normalized protein expression of ZCRB1 reporter constructs with synonymous mutations (HA and β-actin antibodies). Each bar represents the percentage of wild-type ZCRB1-mCherry (WT) expression. (C) Normalized RNA levels of ZCRB1 reporter constructs with synonymous mutations. Neomycin resistance gene was used for normalization of qRT-PCR data. Each bar represents the percentage of wild-type ZCRB1-mCherry construct (WT) mRNA levels. (D) Scheme of full-length HA-tagged ZCRB gene constructs. Position and mutations in poly(A) tracks are indicated. (E) Western blot analysis and normalized protein expression of ZCRB1 gene constructs with synonymous mutations. Each bar represents the percentage of wild-type HA-ZCRB1 (WT) expression. (F) Normalized RNA levels of ZCRB1 gene constructs. Neomycin resistance gene was used for normalization of qRT-PCR data.

  • Fig. 5 Putative mechanisms through which poly(A) tracks exert their function.

    (A) Immunoprecipitation of HA-ZCRB gene constructs using anti-HA magnetic beads. ZCRB1 WT, synonymous (single 411G>A or double 408A>G; 417A>G), nonsense [385G>T, insertion of stop codon before poly(A) track], deletion (423ΔA, equivalent to +1 frameshift), and insertion (423A>AA, equivalent to −1 frameshift) mutant constructs are labeled. (B) Scheme of luciferase constructs used to estimate frameshifting potential for ZCRB1 WT and 411G>A mutant poly(A) tracks. (C) Luciferase levels (activity) from −1, “zero,” and +1 frame constructs of wild-type and G>A mutant ZCRB1 poly(A) tracks are compared. Bars represent the normalized ratio of ZCRB1 G>A and ZCRB1 WT poly(A) tracks, elucidating changes in the levels of luciferase expression in all three frames. (D) Model for function of poly(A) tracks in human genes. Poly(A) tracks lead to three possible scenarios: frameshifting consolidated with NMD, which results in reduced output of wild-type protein; frameshifting with synthesis of both out-of-frame and wild-type protein; and nonresolved stalling consolidated by endonucleolytic cleavage of mRNA and reduction in wild-type protein levels, as in the NGD pathway. Scheme for translation of mRNAs without poly(A) tracks is shown for comparison.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/6/e1500154/DC1

    Fig. S1. Distribution of polyarginine (A) and polylysine (B) runs of different length in several organisms.

    Fig. S2. Expression of HA-X-mCherry reporters in CHO cells.

    Fig. S3. Expression of HA-X-mCherry reporters in Drosophila S2 cells.

    Fig. S4. Expression of HA-X-mCherry reporters from T7-RNA polymerase in vitro transcribed mCherry mRNAs in HDFs.

    Fig. S5. Differential stability of electroporated mRNAs from HA-X-mCherry reporters is translation-dependent.

    Fig. S6. Insertion of polylysine mCherry constructs in the coding sequence results in the same protein reduction and decreased mRNA stability.

    Fig. S7. Expression of HA-tagged hemoglobin (delta chain; HBD) constructs with natural introns in HDF cells.

    Fig. S8. Comparison of usage of AAA in single, double, and triple lysine runs across several organisms.

    Fig. S9. Observed codon usage in all isoforms of human proteins versus expected (based on the proportions 0.44 to 0.56, AAA to AAG for all lysines) in the tracks of four consecutive lysines.

    Fig. S10. Codon distribution in four-lysine tracks in different organisms.

    Fig. S11. Occupancy of ribosomal footprints from three different data sets: (A) region around poly(A) tracks; (B) region around four arginine tracks, all codon combinations together.

    Fig. S12. Sequence conservation of RAS activating-like protein 2 gene (RASAL2) at DNA and protein sequences.

    Fig. S13. Synonymous mutations in mCherry reporter with metadherin [MTDH, Lyric(Lyr)] poly(A) track.

    Fig. S14. Synonymous mutations in mCherry reporter with RASAL2 poly(A) track.

    Fig. S15. Expression analysis of N-terminally HA-tagged and C-terminally GFP-tagged ZCRB1 gene and its synonymous mutants in HDF cells using EVOS FL microscope.

    Fig. S16. Introduction of COSMIC database reported synonymous mutation K447K (1341G>A) in full-length recombinant MTDH gene.

    Fig. S17. Frameshifting efficiency of poly(A) tracks from ZCRB1 wild type (A) and ZCRB G>A mutant (B) measured by luciferase activity.

    Fig. S18. Proportion of mutation types in poly(A) segments versus all mutation types.

    Fig. S19. The normalized distribution of lengths for poly(A) regions identified as 12 A’s allowing for one mismatch up to length 19 in human transcripts.

    Table S1. Statistics of occurrences of transcripts containing poly(A) tracks in different organisms.

    Table S2. Overrepresentation of Gene Ontology terms for 456 genes containing poly(A) tracks in their coding regions up to P value of 0.05.

    Table S3. Table of mRNAs that have intron-exon boundary closer than 50 nucleotides downstream from a stop codon arising from frameshifting over poly(A) tracks.

    Table S4. Peptides arising from possible frameshifting on poly(A) tracks.

    Table S5. Table of genes with mutations within poly(A) region reported in COSMIC database.

    Table S6. Sequences of mCherry inserts.

    Data D1. Analysis of dbSNP database.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Distribution of polyarginine (A) and polylysine (B) runs of different length in several organisms.
    • Fig. S2. Expression of HA-X-mCherry reporters in CHO cells.
    • Fig. S3. Expression of HA-X-mCherry reporters in Drosophila S2 cells.
    • Fig. S4. Expression of HA-X-mCherry reporters from T7-RNA polymerase in vitro transcribed mCherry mRNAs in HDFs.
    • Fig. S5. Differential stability of electroporated mRNAs from HA-X-mCherry reporters is translation-dependent.
    • Fig. S6. Insertion of polylysine mCherry constructs in the coding sequence results in the same protein reduction and decreased mRNA stability.
    • Fig. S7. Expression of HA-tagged hemoglobin (delta chain; HBD) constructs with natural introns in HDF cells.
    • Fig. S8. Comparison of usage of AAA in single, double, and triple lysine runs across several organisms.
    • Fig. S9. Observed codon usage in all isoforms of human proteins versus expected (based on the proportions 0.44 to 0.56, AAA to AAG for all lysines) in the tracks of four consecutive lysines.
    • Fig. S10. Codon distribution in four-lysine tracks in different organisms.
    • Fig. S11. Occupancy of ribosomal footprints from three different data sets: (A) region around poly(A) tracks; (B) region around four arginine tracks, all codon combinations together.
    • Fig. S12. Sequence conservation of RAS activating-like protein 2 gene (RASAL2) at DNA and protein sequences.
    • Fig. S13. Synonymous mutations in mCherry reporter with metadherin MTDH, Lyric(Lyr) poly(A) track.
    • Fig. S14. Synonymous mutations in mCherry reporter with RASAL2 poly(A) track.
    • Fig. S15. Expression analysis of N-terminally HA-tagged and C-terminally GFP-tagged ZCRB1 gene and its synonymous mutants in HDF cells using EVOS FL microscope.
    • Fig. S16. Introduction of COSMIC database reported synonymous mutation K447K (1341G>A) in full-length recombinant MTDH gene.
    • Fig. S17. Frameshifting efficiency of poly(A) tracks from ZCRB1 wild type (A) and ZCRB G>A mutant (B) measured by luciferase activity.
    • Fig. S18. Proportion of mutation types in poly(A) segments versus all mutation types.
    • Fig. S19. The normalized distribution of lengths for poly(A) regions identified as 12 A’s allowing for one mismatch up to length 19 in human transcripts.
    • Table S1. Statistics of occurrences of transcripts containing poly(A) tracks in different organisms.
    • Table S2. Overrepresentation of Gene Ontology terms for 456 genes containing poly(A) tracks in their coding regions up to P value of 0.05.
    • Table S3. Table of mRNAs that have intron-exon boundary closer than 50 nucleotides downstream from a stop codon arising from frameshifting over poly(A) tracks.
    • Table S4. Peptides arising from possible frameshifting on poly(A) tracks.
    • Table S5. Table of genes with mutations within poly(A) region reported in COSMIC database.
    • Table S6. Sequences of mCherry inserts.
    • Data D1. Analysis of dbSNP database.

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