Translational control by lysine-encoding A-rich sequences

Abstract

Regulation of gene expression involves a wide array of cellular mechanisms that control the abundance of the RNA or protein products of that gene. Here we describe a gene-regulatory mechanism that is based on poly(A) tracks that stall the translation apparatus. We show that creating longer or shorter runs of adenosine nucleotides, without changes in the amino acid sequence, alters the protein output and the stability of mRNA. Sometimes these changes result in the production of an alternative "frame-shifted" protein product. These observations are corroborated using reporter constructs and in the context of recombinant gene sequences. Approximately two percent of genes in the human genome may be subject to this uncharacterized, yet fundamental form of gene regulation. The potential pool of regulated genes encodes many proteins involved in nucleic acid binding. We hypothesize that the genes we identify are part of a large network whose expression is fine-tuned by poly(A)-tracks, and we provide a mechanism through which synonymous mutations may influence gene expression in pathological states.

Keywords: Lysine; gene regulation; mRNA stability; ribosome stalling; synonymous mutations.

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.