Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Sequences required for transcription termination at the intrinsic lambdatI terminator.

The lambdatI terminator is located approximately 280 bp beyond the lambdaint gene, and it has a typical structure of an intrinsic terminator. To identify sequences required for lambdatI transcription termination a set of deletion mutants were generated, either from the 5' or the 3' end onto the lambdatI region. The termination efficiency was determined by measuring galactokinase (galK) levels by Northern blot assays and by in vitro transcription termination. The importance of the uridines and the stability of the stem structure in the termination were demonstrated. The nontranscribed DNA beyond the 3' end also affects termination. Additionally, sequences upstream have a small effect on transcription termination. The in vivo RNA termination sites at lambdatI were determined by S1 mapping and were located at 8 different positions. Processing of transcripts from the 3' end confirmed the importance of the hairpin stem in protection against exonuclease.

Overexpression of mutant dystrophin Dp71[INCREMENT]78-79 stimulates cell proliferation.

Dp71 dystrophin is the main DMD gene product expressed in the central nervous system. Experiments using PC12 cells as a neuronal model have shown that Dp71 isoforms are involved in differentiation, adhesion, cell division, and nuclear architecture. To contribute to the knowledge of Dp71 domains function, we previously reported the isolation and partial characterization of the dystrophin Dp71[INCREMENT]78-79 (a mutant that lacks exons 71, 78, and 79), which stimulates the neuronal differentiation of PC12-C11 clone. In this article, we generated a doxycycline (Dox)-inducible expression system in PC12 Tet-On cells (B10 cells) to overexpress and control the transcription of Dp71[INCREMENT]78-79. Western blotting and confocal microscopy showed an increase in the amount of Dp71[INCREMENT]78-79 (217±75-fold) with the addition of Dox to growth medium. Cell proliferation assays and morphometric analyses demonstrated that Dp71[INCREMENT]78-79 increases the growth rate of B10 cells and reduces the nerve growth factor-neuronal differentiation. Western blotting analysis revealed an upregulation in the expression of proliferating cell nuclear antigen, focal adhesion kinase, and ß-dystroglycan in B10 cells compared with control cells. Our results show that the inducible expression of Dp71[INCREMENT]78-79 increases the growth rate of PC12 Tet-On cells, suggesting a role of this protein in cell proliferation.

Identification of Dp71e, a new dystrophin with a novel carboxy-terminal end.

Several dystrophin Dp71 isoforms have previously been described and can be grouped into two subfamilies (Dp71d or Dp71f) depending upon the splicing of exon 78. As a consequence of this splicing, each group has a carboxy-terminal end with a unique amino acid composition; this composition imparts specific characteristics with respect to subcellular localization and interactions with particular members of the dystrophin-associated proteins (DAPs) complex. We have discovered a new alternative splicing event at the 3' region of the Dp71 transcript. This spliced region has a unique sequence that codes for 10 amino acids and prevents the translation of exons 78 and 79. This novel Dp71 isoform is called Dp71e and is expressed in undifferentiated cells and during nerve growth factor-induced differentiation of PC12 cells. Interestingly, Dp71e mRNA and protein expression increase during PC12 cell differentiation mediated by NGF. This new Dp71 isoform is also expressed in rat organs and in human cell lines.

α-Enolase binds to RNA.

To detect proteins binding to CUG triplet repeats, we performed magnetic bead affinity assays and North-Western analysis using a (CUG)(10) ssRNA probe and either nuclear or total extracts from rat L6 myoblasts. We report the isolation and identification by mass spectrometry and immunodetection of α-enolase, as a novel (CUG)n triplet repeat binding protein. To confirm our findings, rat recombinant α-enolase was cloned, expressed and purified; the RNA binding activity was verified by electrophoretic mobility shift assays using radiolabeled RNA probes. Enolase may play other roles in addition to its well described function in glycolysis.