Neural Isoforms of Agrin Are Generated by Reduced PTBP1-RNA Interaction Network Spanning the Neuron-Specific Splicing Regions in AGRN.

Agrin is a heparan sulfate proteoglycan essential for the clustering of acetylcholine receptors at the neuromuscular junction. Neuron-specific isoforms of agrin are generated by alternative inclusion of three exons, called Y, Z8, and Z11 exons, although their processing mechanisms remain elusive. We found, by inspection of splicing cis-elements into the human AGRN gene, that binding sites for polypyrimidine tract binding protein 1 (PTBP1) were extensively enriched around Y and Z exons. PTBP1-silencing enhanced the coordinated inclusion of Y and Z exons in human SH-SY5Y neuronal cells, even though three constitutive exons are flanked by these alternative exons. Deletion analysis using minigenes identified five PTBP1-binding sites with remarkable splicing repression activities around Y and Z exons. Furthermore, artificial tethering experiments indicated that binding of a single PTBP1 molecule to any of these sites represses nearby Y or Z exons as well as the other distal exons. The RRM4 domain of PTBP1, which is required for looping out a target RNA segment, was likely to play a crucial role in the repression. Neuronal differentiation downregulates PTBP1 expression and promotes the coordinated inclusion of Y and Z exons. We propose that the reduction in the PTPB1-RNA network spanning these alternative exons is essential for the generation of the neuron-specific agrin isoforms.

Zonisamide upregulates neuregulin-1 expression and enhances acetylcholine receptor clustering at the in vitro neuromuscular junction.

Decreased acetylcholine receptor (AChR) clustering compromises signal transmission at the neuromuscular junction (NMJ) in myasthenia gravis, congenital myasthenic syndromes, and motor neuron diseases. Although the enhancement of AChR clustering at the NMJ is a promising therapeutic strategy for these maladies, no drug is currently available for this enhancement. We previously reported that zonisamide (ZNS), an anti-epileptic and anti-Parkinson's disease drug, enhances neurite elongation of the primary spinal motor neurons (SMNs). As nerve sprouting occurs to compensate for the loss of AChR clusters in human diseases, we examined the effects of ZNS on AChR clustering at the NMJ. To this end, we established a simple and quick co-culture system to reproducibly make in vitro NMJs using C2C12 myotubes and NSC34 motor neurons. ZNS at 1-20 µM enhanced the formation of AChR clusters dose-dependently in co-cultured C2C12 myotubes but not in agrin-treated single cultured C2C12 myotubes. We observed that molecules that conferred responsiveness to ZNS were not secreted into the co-culture medium. We found that 10 µM ZNS upregulated the expression of neuregulin-1 (Nrg1) in co-cultured cells but not in single cultured C2C12 myotubes or single cultured NSC34 motor neurons. In accordance with this observation, inhibition of the Nrg1/ErbB signaling pathways nullified the effect of 10 µM ZNS on the enhancement of AChR clustering in in vitro NMJs. Although agrin was not induced by 10 µM ZNS in co-cultured cells, anti-agrin antibody attenuated ZNS-mediated enhancement of AChR clustering. We conclude that ZNS enhances agrin-dependent AChR-clustering by upregulating the Nrg1/ErbB signaling pathways in the presence of NMJs.

Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells.

Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA-binding protein, which is highly expressed in skeletal muscle. Abnormally expanded CUG-repeats in the DMPK gene cause myotonic dystrophy type 1 (DM1) by sequestration of MBNL1 to nuclear RNA foci and by upregulation of another RNA-binding protein, CUG-binding protein 1 (CUGBP1). We previously reported that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone, upregulates MBNL1 expression in DM1 mouse model by demethylation of MeR2, an enhancer element in Mbnl1 intron 1. NSAIDs inhibit cyclooxygenase (COX), which is comprised of COX-1 and COX-2 isoforms. In this study, we screened 29 NSAIDs in C2C12 myoblasts, and found that 13 NSAIDs enhanced Mbnl1 expression, where COX-1-selective NSAIDs upregulated Mbnl1 more than COX-2-selective NSAIDs. Consistently, knockdown of COX-1, but not of COX-2, upregulated MBNL1 expression in C2C12 myoblasts and myotubes, as well as in myotubes differentiated from DM1 patient-derived induced pluripotent stem cells (iPSCs). Luciferase assay showed that COX-1-knockdown augmented the MeR2 enhancer activity. Furthermore, bisulfite sequencing analysis demonstrated that COX-1-knockdown suppressed methylation of MeR2. These results suggest that COX-1 inhibition upregulates Mbnl1 transcription through demethylation of the MeR2 enhancer. Taken together, our study provides new insights into the transcriptional regulation of Mbnl1 by the COX-1-mediated pathway.

Draft Genome Sequence of Grammothele lineata SDL-CO-2015-1, a Jute Endophyte with a Potential for Paclitaxel Biosynthesis.

Grammothele lineata strain SDL-CO-2015-1, a basidiomycete fungus, was identified as an endophyte from a jute species, Corchorus olitorius var. 2015, and found to produce paclitaxel, a diterpenic polyoxygenated pseudoalkaloid with antitumor activity. Here, we report the draft genome sequence (42.8 Mb with 9,395 genes) of this strain.