The Spinal Muscular Atrophy Disease Protein SMN Is Linked to the Golgi Network

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From: PLoS ONE(Vol. 7, Issue 12)
Publisher: Public Library of Science
Document Type: Article
Length: 7,462 words
Lexile Measure: 1470L

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Author(s): Chen-Hung Ting 1 , * , Hsin-Lan Wen 1 , Hui-Chun Liu 1 , 2 , Hsiu-Mei Hsieh-Li 3 , Hung Li 1 , Sue Lin-Chao 1 , *


SMN protein deficiency causes human spinal muscular atrophy (SMA), which is characterized as an autosomal recessive neurodegenerative disorder [1]-[4]. SMN is produced by SMN1 and its paralog gene, SMN2 , which is nearly identical to SMN1 with the exception of a C to T transition in exon 7 that causes alternative SMN2 exon 7 splicing resulting in the exclusion of exon 7 in over 80% of SMN2 transcripts and expression of an unstable SMN protein [5]-[7]. SMN2 cannot compensate for the loss of SMN1 ; however, genetic evidence demonstrates that SMN2 copy number influences disease severity, supporting the notion that SMN2 could serve as a disease modifier [8]-[11].

SMN is ubiquitously expressed and is involved in snRNP assembly [12]-[14]. Additionally, SMN has been shown to move along the axon in granules [15], [16] and to form complexes that lack most of the interacting partners involved in snRNP assembly in motor neurons [17], [18]. Motor neurons lacking SMN show some axonal abnormalities [19]-[21] such as growth cone defects [19], suggesting a specific role of SMN granules in axons. SMN is known to interact with the [alpha]-coatomer (Cop-[alpha]) [22] that has been shown to mediate vesicle trafficking between the Golgi compartments [23]; however, a direct link between the SMN granule and the Golgi apparatus has not been shown.

Herein, we report that cytoplasmic SMN can be detected in the Golgi-enriched fractions. Time-lapse studies show that SMN granules associate with the Golgi apparatus and move like a regulated secretory granule. Global blockade of granule secretion from the trans -Golgi network resulted in decreased levels of SMN in neurites and a decrease in the size of growth cones. SMN granules retained in the Golgi apparatus were found in Cop-[alpha] knockdown cells, suggesting that Cop-[alpha] is involved in SMN granule secretion from the Golgi. Our work links SMN granules with the Golgi apparatus and implicates them in Golgi-mediated SMN granule transport.

Materials and Methods

Antibodies, Chemicals, Small Interfering RNAs, and Plasmids

Anti-Gemin2 (clone 2E17) was purchased from Sigma. Anti-SMN (clone 2B1) antibody was from BD Biosciences. Antibodies against Gm130 (clone EP892Y), Golgin-97, and Calnexin were from Abcam, and the antibody against Tgn38 was from Novus Biologicals. Anti-Syncrip and anti-COP[alpha] antibodies were from AVIVA Systems Biology. Antibodies specific for histone H1 (clone AE-4), [alpha]-tubulin (clone DM1A), [beta]III -tubulin, and ChAT were purchased from Millipore. Anti-U2af65 and Alexa Fluor fluorescence dye-conjugated secondary antibodies (Phalloidin), as well as scrambled siRNA and siRNAs against mSmn (siRNA1:5'-UAAAGUCAAUGGACGUAAUAGUAGC-3'; siRNA2:5'-UACUAUUAGCUACUUCACAGGUCGG-3'; siRNA3:5'-AAAUGUCAGAAUCAUCACUCUGGCC-3'; siRNA4:5'-UGGCUAAGUGGUGUCGUCAUCAGCA-3') were purchased from Invitrogen. Cop-[alpha] SMART-pool siRNAs and scrambled siRNAs were purchased from Thermo Scientific Dharmacon (<named-content content-type="gene" xlink:type="simple">5'-GGAACAAGCCCAACGCAAA-3'</named-content>; 5'-GAACAGUACCUACGACCUA-3'; 5'-GGUUUGGGUUGCUCGGAAU-3'; 5'-CAAGUGAAGAUCUGGCGUA-3'). The pDsRed-monomer-[beta]1,4-galactosyltransferase ([beta]1,4-Gal-T) plasmid was purchased from Clontech. For constructing the mCherry-tagged SMAD anchor for receptor activation (SARA)-FYVE domain (mCherry-FYVE), the domain was amplified from pCMV5B-FLAG-SARA (Addgene plasmid 11738) [24] by PCR using a specific primer pair (forward:...

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Gale Document Number: GALE|A477075778