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大学・研究所にある論文を検索できる 「Neural Isoforms of Agrin Are Generated by Reduced PTBP1−RNA Interaction Network Spanning the Neuron−Specific Splicing Regions in AGRN」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。
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Neural Isoforms of Agrin Are Generated by Reduced PTBP1−RNA Interaction Network Spanning the Neuron−Specific Splicing Regions in AGRN

Samira, Bushra 名古屋大学

2023.10.05

概要

主論文の要旨

Neural Isoforms of Agrin Are Generated by Reduced
PTBP1-RNA Interaction Network Spanning the
Neuron-Specific Splicing Regions in AGRN
AGRN遺伝子の神経特異的スプライシング領域に広がる
PTBP1-RNAネットワークが縮小することで、
Agrinの神経型アイソフォームが生成される

名古屋大学大学院医学系研究科
先端応用医学講座

総合医学専攻

神経遺伝情報学分野

（指導：大野 欽司
Samira Bushra

教授）

Background
Agrin is an extracellular matrix protein, expressed as multiple isoforms in diverse
tissues that play roles various cellular processes. Neuron-spec ific agrin isoforms are
generated through alternative splicing of AGRN pre-mRNA and are exclusively capable
of inducing acetylcholine receptor (AChR) clustering. 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 (Figure 1A). The 12-nt alternative
cassette Y exon encodes four amino acids that confer the interactions with heparin and
dystroglycan. Alternative inclusion and skipping of the Y exon generate the Y4 and Y0
isoforms, respectively (Figure 1A). Inclusion of Z8, Z11, and both Z8 and Z11 exons,
generate the Z8, Z11, and Z19 isoforms, respectively, which are crucial for AChR
clustering. Splicing of Y, Z8, and Z11 exons is coordinately re gulated in rat, resulting
in abundant expression of Y4Z8 and Y4Z19 isoforms and minimal expression of other
isoforms (Y4Z11 and Y0Z0/8/11/19) in neural tissues. In contrast, the Y0Z0 isoform is
widely expressed in non-neural tissues. Despite the essential r ole of the neural isoforms
of agrin in AChR clustering, the splicing regulating mechanism(s) to generate neuronspecific isoforms of agrin remain to be disclosed. In the present study, we dissected the
underlying mechanisms that regulate coordinate regulation of AGRN alternative splicing.
Methods
We employed RT-PCR, siRNA-mediated knockdown, site directed mutagenesis, cDNA
overexpression, tethered function assay, immunoblotting, in vitro binding assay and bioinformatic
analysis of eCLIP-seq and RNA-seq data to dissect the molecular mechanism that regulate
AGRN alternative splicing.
Results
We first analyzed the splicing patterns of Y and Z exons of AGRN in various human tissues
using RT-PCR and found Y and Z exons were included only in the human brain (Figure 1B).
Nested RT-PCR analysis from Y+/Y transcripts demonstrated coordinated splicing of the Y
exon with the Z exons (Figure 1B). Next, we induced neuronal differentiation of the SH-SY5Y
human neuroblastoma cell line and RT-PCR analysis showed that the inclusion of Y and Z
exons was gradually increased with neuronal differentiation (Figure 1C-G). These results
indicate that the Y, Z8, and Z11 exons are coordinately included in differentiated human
neuronal cells and in the human brain.
To identify splicing cis-element/s and cognate splicing trans-factor/s involved in the
alternative splicing of AGRN, we analyzed distribution of all possible di-nucleotide motifs
from exons 30 to 37 in the human AGRN gene, whereby exons 31, 35, and 36 are Y4, Z8, and
Z11 exons, respectively. We observed the enrichment of the CU/UC motif around Y, Z8, and

cis
AGRN

AGRN

AGRN

AGRN

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