Non-ATG-initiated translation directed by microsatellite expansions

Abstract

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.

Fig. 1.

Non-ATG translation in HEK293T cells. (A) Immunoblot (Right) from A8-transfected cells (Left) with or without the ATG start codon. (B) (Upper) Modified A8 constructs with the 6X STOP cassette and 3′ tags in each frame [A8(*KKQ_EXP)-3Tf1]. (Lower) Immunoblots of lysates with or without proteinase K, DNase I, RNase I (Left) and of cells treated with cycloheximide (CHX) (Right). An ATG start codon in the polyGln frame variably results in the generation of a second, higher molecular weight band, and this sequence change also can affect polyAla migration and relative levels of polySer. (C) Immunoblots of A8(*KKQ_EXP)-3Tf1, A8(*KKQ_EXP)-3Tf2, and A8(*KKQ_EXP)-3Tf3 lysates show that polyAla, polyGln, and polySer are expressed at relatively high, intermediate, and low levels, respectively. The additional lower molecular weight polyGln band expressed from A8(*KKQ_EXP)-3Tf2 is caused by sequence heterogeneity after repeat contraction in Escherichia coli. (D) IF staining of polyGln (α-His/cy3), polyAla (α-HA/cy5), and polySer (α-FLAG/FITC).

Fig. 2.

Protein labeling and MS. (A) Protein blot (Upper) and fluorograph (Lower) of [³H]-Gln, [³H]-Ala, or [³H]-Ser after immunoprecipitation with α-HA of cells transfected with A8(*KKQ_EXP)-3Tf1, A8(*KKQ_EXP)-3Tf2, A8(*KKQ_EXP)-3Tf3, or empty vector. (B) (Upper) Diagram of IntR(GCA_EXP)-3T construct with CGCGCG interruption. (Lower) Predicted sequence of polyAla protein with arginine interruption. (C) Preparative deep-purple gel showing slices digested with trypsin for MS. (D) N-terminal polyAla peptides identified by MS with varying numbers of alanine. (E) Representative spectrum of N-terminal peptide AAAAAAAAAR with matched b-ions (red) and y-ions (blue).

Fig. 4.

Repeat length, sequence motif, and toxicity. (A) Immunoblots of lysates after transfection with A8(*KKQ_EXP)-3Tf1 or A8(*KMQ_EXP)-3Tf1 with varying repeat lengths. (B) (Upper) Triple-tagged constructs containing a CAA or CAG repeat tract with or without ATG. (Lower) Corresponding immunoblot. (C) (Left) Relative levels of annexin-V–positive N2a cells transfected with [ATT(CAG₁₀₅)-3T, ATG(CAG₁₀₅)-3T] compared with polyGln only [ATG(CAA₉₀)-3T] and negative [ATT(CAA₉₀)-3T] controls. (Right) Corresponding immunoblots. **P < 0.01; ***P < 0.001].

Fig. 3.

Polysome-associated transcripts and RNA transfections. (A) (CAG_EXP)-3T constructs with or without an ATG initiation codon. (B) Polyribosome profiles from transfected cells. (Upper) OD 254. (Lower) RNA blots with relative levels of CAG transcripts. (C) (Upper) 5′ RACE and RT-PCR strategy used to characterize (+)AUG and (−)AUG transcripts from polysome fraction #6. (Lower) Table summarizing sequencing results from cloned RT-PCR products. (D) In vitro transcription and RNA transfection into HEK293T cells. (Upper) Constructs used to produce capped, polyadenylated mRNAs that extend from the T7 promoter to the PvuII site (P). (Lower) Immunoblots following RNA transfections.

Fig. 5.

RAN translation in the presence of an ATG-initiated ORF. (A) Constructs with 5′ V5 tag in glutamine frame and 3′ tags. (B) Corresponding protein blots. (C) Protein blots after immunoprecipitation with antibodies to 3′ epitope tags in polyGln (α-His), polyAla (α-HA), and poly-Ser (α-Flag) frames probed for the 5′ epitope tag with α-V5 (Upper) or 1C2, α-HA, α-Flag (Lower).

Fig. 6.

In vivo evidence for RAN-translated SCA8-polyAla and DM1-polyGln. (A) ATXN8 ATG-polyGln ORF and putative non-ATG SCA8_GCA-Ala protein. Underlined peptides used for antibody generation. (B) Antibody validation: α-SCA8_GCA-Ala detection of recombinant protein in A8(*KMQ_EXP)-endo transfected cells by protein blot (Left) and IF (Right). (C) Immunohistochemical staining of SCA8 and control mouse cerebellum (FVB) using α-SCA8_GCA-Ala. (D) In SCA8 human samples, α-SCA8_GCA-Ala antibody shows consistent and specific staining (red-cy3) of surviving human SCA8 cells but not control Purkinje cells. Colabeling with α-PKCγ antibody (cy5, yellow) independently stains Purkinje cells and confirms their presence in both samples. (E) (Upper) DM1 CAG antisense transcript. (Lower) Predicted non-ATG–initiated polyGln protein. Underlined peptide used for antibody generation. (F) Antibody validation: α-DM1_CAG-Gln detects recombinant protein with endogenous DM1 polyGln C-terminus (CAG_EXP-DM1-3’) by protein blot (Left) and IF (Right). (G) IF using α-DM1_CAG-Gln (cy3, red) detects DM1_CAG-Gln protein in cardiomyocytes of DM1 mice with 55 (DM55), and >1,000 (DMSXL) CTGs but not in cardiomyocytes of control (WT) mice. (H) Staining with α-DM1_CAG-Gln (cy3, red) in DM1 but not in control (WT) myoblasts.