Splicing variants of porcine synphilin-1

Abstract

Parkinson's disease (PD), idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB) in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90%) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation.

Keywords: LB, Lewy body; Lewy body; ORF, open reading frame; Parkinson's disease; Pig; SNCAIP, α-synuclein interacting protein; Synphilin-1; α-Synuclein.

Fig. 1

Schematic presentation of the Synphilin-1 protein. The characteristic domains of synphilin-1 are indicated by their respective names.

Fig. 2

Multiple alignment of the porcine synphilin-1 and with synphilin-1 sequences from human and mouse. Alignments of sequences were performed using the Clustal W program on EBI WWW molecular biology server. The numbers represent the position of the amino acids in the respective protein sequences. Identical amino acid residues in all sequences are indicated by asterisks. Arrowheads indicate intron–exon boundaries. The abbreviations for species acronyms and corresponding accession numbers of the sequences used for the alignment are: Hs = Homo sapiens (NM_005460); Ss = Sus scrofa (NM_001105053); Mm = Mus musculus; (NM_026408).

Fig. 3

Splice variants of porcine SNCAIP. Amino acid sequence alignment of porcine synphilin-1 and a splice variant, synphilin-1tv1, hereof (A). The synphilin-1tv1 is lacking exon 2–exon 5. Also, a second variant of synphilin-1, synphilin-1tv2, was identified by the cloning (B). This splice variant is homologous with human transcript variant synphilin-1g.

Fig. 4

Expression of porcine SNCAIP mRNA determined by RNAseq. Relative abundance of Synphilin-1 and Synphilin-1tv1 in FPKM units. The tissues presented are: hypothalamus (HYP), lung (LUN), frontal cortex (FCO), occipital cortex (OCC), cerebellum (CBE), spleen (SPL), heart (HEA), kidney (KID), liver (LIV) and musculus longissimus dorsii (LDO). The error bars represent the biological variation between the two animals employed.