Expansion of a CUG trinucleotide repeat in the 3' untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts

Abstract

Expansion of a CTG trinucleotide repeat in the 3' untranslated region (UTR) of DMPK, the gene encoding myotonic dystrophy protein kinase, induces the dominantly inherited neuromuscular disorder myotonic dystrophy (DM). Transcripts containing the expanded trinucleotide are abundant in differentiated cultured myoblasts, and they are spliced and polyadenylylated normally. However, mutant transcripts never reach the cytoplasm in these nonmitotic cells; instead, they form stable clusters that are tightly linked to the nuclear matrix, which can prevent effective biochemical purification of these transcripts. In DM patients, reduced DMPK protein levels, consequent to nuclear retention of mutant transcripts, are probably a cause of disease development. Formation of nuclear foci is a novel mechanism for preventing transcript export and effecting a loss of gene function.

Figure 1

Northern analysis of DMPK and myogenin transcription in normal and MyoD-infected fibroblasts. Lane 1, fibroblast RNA; lanes 2–5, myoblast RNA after 0, 1, 2, or 3 days of differentiation; 10 μg of total RNA per lane. (A) Blot probed with DM3′A. (B) Blot probed with myogenin cDNA. (C) Ethidium bromide staining of gel prior to transfer.

Figure 2

Northern analysis of DMPK transcripts in representative normal and myotonic myoblasts. Lanes 1 and 2, normal myoblast RNA; lanes 3, 4, and 5, myotonic myoblast RNA; probed with DM3′A. When multiple mutant bands were detected, all were included in calculating transcript abundance.

Figure 3

Comparison of nuclear and cytoplasmic DMPK RNAs isolated from control and myotonic cells. RNA for each nuclear (N)/cytoplasmic (C) pair was extracted from the same cells. Northern blots were probed with DM5′A/DM5′B. (A) Lanes 1–4, control myoblasts; lanes 5–12, myotonic myoblasts. Ethidium bromide staining prior to transfer is shown on the right. (B) Nuclear and cytoplasmic RNA from uninfected DM fibroblasts. (C) RNA isolated from DM myoblast nuclei by CsCl ultracentrifugation (lane 1) or acid guanidinium thiocyanate/phenol/chloroform (lane 3). Cytoplasmic RNAs (lanes 2 and 4) were purified by CsCl ultracentrifugation of cytoplasmic fractions.

Figure 4

Distribution of triplet repeat transcripts in myoblasts and dividing cells. Fixed cells on coverslips were hybridized with cy3-labeled CAG-30 and counterstained with DAPI. Spots represent signal from the trinucleotide repeat. (A–E, ×225; F, ×300.) (A) Analog photomicrograph. (B–F) Digital images, generated by mathematically removing out-of-focus light from images captured by a CCD camera. Each digital image is a restored optical section through the cell(s). (A and B) DM myoblasts. (C) Normal myoblasts. (D) DM myoblasts from a minimally affected patient with approximately 150 CTG repeats. (E) DM myoblasts treated with DNase I and extracted with (NH₄)₂SO₄. The expanded triplet repeat molecules remained in the nuclear matrix. The lack of DAPI staining indicates that the DNA has been removed. (F) Dividing fibroblasts from the same patient as in A. In late anaphase (possibly telophase), no signal was detected in the newly reformed nucleus, suggesting that transcript foci have diffused within the cytoplasm. (Left) Dividing cell without DAPI stain. (Right) DAPI shows the condensed DNA typical of anaphase.

Figure 5

(A) Northern analysis of association of DMPK transcripts with the nuclear matrix. Some wt DMPK message is present in the cytoplasmic (lanes 1 and 5) and cytoskeletal (lanes 2 and 6) fractions, and a small amount is chromatin associated (lanes 3 and 7). All the mutant transcripts and 40–50% of the wt transcripts were retained with the nuclear matrix (lanes 4 and 8). Lanes 1–4, control cells; lanes 5–8, myotonic cells; probed with DM5′A/DM5′B. (B) Northern analysis of polyadenylylation of wt and mutant DMPK transcripts. Ten micrograms of total (T) RNA and the flowthrough (FT) and poly(A)⁺ (A+) fractions isolated from 20 μg of total RNA were blotted and probed with DM5′A/DM5′B. Lanes 1–3, control cells; lanes 4–9, DM cells.

Figure 6

Northern analysis of oligonucleotide-directed cleavage of DMPK transcripts with RNase H. Undigested (lanes 1–3) and digested (lanes 4–6) total RNA, 15 μg per lane; probed with DM5′A, DM5′B, and DM3′B. Lanes 1 and 4, wt2; lanes 2 and 5, DM4; and lanes 3 and 6, DM1.

Figure 7

Northern analysis of DMPK mRNA stability. Nuclear (N) and cytoplasmic (C) RNA was probed with DM3′A. (A) DM cells (DM4). (B) Control cells (wt3).