Localization of ␤-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3 untranslated region of the mRNA, including a 54-nucleotide element which has been termed the "zipcode" (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize ␤-actin mRNA.

Abstract. We have analyzed the intracellular localization of transcripts from the myotonin protein kinase (Mt-PK) gene in fibroblasts and muscle biopsies from myotonic dystrophy patients and normal controls. In affected individuals, a trinucleotide expansion in the gene results in the phenotype, the severity of which is proportional to the repeat length. A fluorochromeconjugated probe (10 repeats of CAG) hybridized specifically to this expanded repeat. Mt-PK transcripts containing CTG repeat expansions were detected in the nucleus as bright foci in DM patient fibroblasts and muscle biopsies, but not from normal individuals. These foci represented transcripts from the Mt-PK gene since they simultaneously hybridized to fluorochrome-conjugated probes to the 5'-end of the Mt-PK mRNA. A single oligonucleotide probe to the repeat and the sense strand each conjugated to different tluorochromes revealed the gene and the transcripts simultaneously, and indicated that these focal concentrations (up to 13 per nucleus) represented predominately posttranscriptional RNA since only a single focus contained both the DNA and the RNA. This concentration of nuclear transcripts was diagnostic of the affected state, and may represent aberrant processing of the RNA.

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes for in situ hybridization, we observed distinct localization patterns for beta- and gamma-actin within cultured cerebrocortical neurons. beta-Actin protein was highly enriched within growth cones and filopodia, in contrast to gamma-actin protein, which was distributed uniformly throughout the cell. beta-Actin protein also was shown to be peripherally localized after transfection of beta-actin cDNA bearing an epitope tag. beta-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike gamma-actin mRNAs, which were restricted to the cell body. The rapid localization of beta-actin mRNA, but not gamma-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolution in situ hybridization and image-processing methods, we showed that the distribution of beta-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. beta-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of beta-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.

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.Myotonic dystrophy (DM), an autosomal dominant neuromuscular disorder, is due to the extreme expansion of a trinucleotide (CTG) repeat in the 3Ј untranslated region (UTR) of the gene encoding DM protein kinase, DMPK (1-3). Mutant DMPK alleles contain up to several thousand triplets, rather than the normal 5-35. Since the expanded repeat is not translated, and hence should not directly alter the gene's protein kinase product, disease models based on overexpression (4) or underexpression (5) of the kinase have been proposed. Most recent studies (6-8) support a loss-of-function model for DM development; however, the cause of DMPK deficiency has been controversial.Loss of DMPK function could result from a novel feature of mutant DMPK transcripts that was revealed by in situ hybridization. Taneja et al. (9) demonstrated that mutant DMPK transcripts form nuclear foci in DM fibroblasts and muscle; foci were not detected in control fibroblasts or muscle biopsies. It was postulated that these nuclear foci might contribute to DM pathogenesis, perhaps by disrupting transport of mRNA from DMPK and͞or other genes to the cytoplasm. However, the division of fibroblasts in culture provided a potential pathway for release of nuclear RNA into the cytoplasm, preventing conclusive testing of this hypothesis. Mutant DMPK transcripts were in fact detected within the cytoplasm of DM fibroblasts; the localization and cytoskeletal association of these transcripts did not differ from those of wild-type (wt) transcripts.The pathology of DM is most evident in differentiated muscle tissue. We have therefore examined DMPK transcription and transcript processing in control and DM cultured myoblasts, generated by MyoD retroviral infections of fibroblasts. Since differentiated myoblasts are arrested in G 0 of the cell cycle, we could also test the hypothesis that the repeat expansion interferes with nuclear export of transcripts. Normal (wt) and mutant DMPK transcripts were analyzed by both Northern blotting and in situ hybridization.Our analyses revealed that mutant DMPK transcripts were abundant in myoblasts, but could not contribute to kinase production, as the transcripts were...