Increasing the Coding Potential of Genomes Through Alternative Splicing: The Case of PARK2 Gene

Abstract

The completion of the Human Genome Project aroused renewed interest in alternative splicing, an efficient and widespread mechanism that generates multiple protein isoforms from individual genes. Although our knowledge about alternative splicing is growing exponentially, its real impact on cellular life is still to be clarified. Connecting all splicing features (genes, splice transcripts, isoforms, and relative functions) may be useful to resolve this tangle. Herein, we will start from the case of a single gene, Parkinson protein 2, E3 ubiquitin protein ligase (PARK2), one of the largest in our genome. This gene is implicated in the pathogenesis of autosomal recessive juvenile Parkinsonism and it has been recently linked to cancer, leprosy, autism, type 2 diabetes mellitus and Alzheimer's disease. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification and protein diversity in tissues and cells. This review will provide an update of all human PARK2 alternative splice transcripts and isoforms presently known, and correlate them to those in rat and mouse, two common animal models for studying human disease genes. Alternative splicing relies upon a complex process that could be easily altered by both cis and trans-acting mutations. Although the contribution of PARK2 splicing in human disease remains to be fully explored, some evidences show disruption of this versatile form of genetic regulation may have pathological consequences.

Keywords: Alternative splicing; PARK2; Protein isoforms; Splice expression patterns.; Splice variants; mRNA.

Fig. (1)

The alternative splicing mechanism.The spliceosome machinery (U1, U2, U4, U5 and U6) assembles on the nascent pre-mRNA. The conserved sequences that enable recognition of the mRNA by the spliceosome are: the 5’ splice site (GU), the 3’ splice site (AG), the branch point and the polypyrimidine tract (PPT). In E complex, U1 forms a base-pairing interaction with the 5’-splice site, whereas U2 base-pairs with the branch-point. Then, a tri-snRNP complex containing U4, U5 and U6 associates with the forming spliceosome, removing U1 and U4 (C complex). These steps allow the two transesterification reactions and join the exons.

Fig. (2)

Human PARK2 gene and exonic structure of alternative splice variants. A: Exonic and intronic organization of human PARK2 gene. Exons are represented as red bars. The size of introns (black line) is proportional to their length. B: Exon organization map of the 21 human PARK2 splice variants currently known. Exons are represented by shaded boxes (gray for non coding sequence, white for coding sequence, blue for UBQ domain and green for IBR domains) with a size proportional to their length. The first (1) and last (17) exons are represented entirely, although their sequence is partial in some variants (H1-H5, H7-H21).

Fig. (3)

Rat Park2 gene and exonic structure of alternative splice variants. A: Exons and introns organization of rat Park2 gene. B: Exon organization map of the 20 rat Park2 splice variants currently known. For details, see Fig. 2. Exon 1 and exon 20 sequences are partial in some variants (R1-R13).

Fig. (4)

Mouse Park2 gene and exonic structure of splice variants. A: Exons and introns organization of mouse Park2 gene. B: Exon organization map of the 9 mouse Park2 splice variants currently known. For details, see Fig. 2. Exon 1 and exon 15 sequences are partial in some variants (M5-M9).

Fig. (5)

Exonic structures of human, rat and mouse PARK2 genes. Homologous sequences are shown in the same column. Green boxes represent the known exons of PARK2 genes. Yellow boxes represent homologous sequences that could be potentially expressed, since they are provided with splice sites (AG/GT). Red boxes represent homologous sequences without splice sites and, therefore, not expressible. Numbers in the top of the figure indicate percentages of homology between human and rat exons, while those at the bottom denote percentages of homology between human and mouse exons.

Fig. (6)

Predicted molecular architecture of PARK2 isoforms.PARK2 isoforms contain one or more of the following domains: an N-terminal Ubiquitin-like domain (UBQ, in blue) and one or two In Between Ring finger domains (IBR, in green). The code identifier of each isoform, reported on their left, corresponds to that of the encoding splice variants listed in (Tables 1, 3 and 4).

Fig. (7)

Differential expression of Park2 transcript variants in rat brain and isolated cells. Single-stranded cDNAs from adult rat brain, rat cortical neurons, rat cerebellar granule cells and rat cortical type I astrocytes mRNAs was PCR amplified with primers flanking the start and the stop codon of Park2. The resulting splicing patterns clearly show a regional and cellular differential expression among different rat neuronal cells, producing spatial and functional diversification. Marker length (bp) is shown on the left. Known rat splice variants with a length between 1200 and 2000 bp are shown on the right (for codes, see Table 3). All the other products have not yet been cloned and remain unknown.

Fig. (8)

Differential expression of Park2 isoforms in rat cortical neurons and type I astrocytes. A: Western Blot analysis of Park2 isoforms in rat neurons (lane 1) and type I astrocytes (lane 2). The assay was performed using a rabbit anti-Park2 polyclonal antibody (AB5112, Millipore) as previously described  ADDIN EN.CITE  ADDIN EN.CITE.DATA [49]. Expected molecular weights of Park2 isoforms potentially recognized by this antibody are drawn on lane 3. The very intense immunoreactive band of about 51 kDa in cortical neurons overlaps with the expected molecular weight of several isoforms (R1, R2, R4-R10, R13-R15, and R19). Instead, type I astrocytes express a faint band of 51 kDa which may correspond to R1 isoform, and a 65 kDa band that corresponds to a still uncharacterized variant. It should be noted that the antibody used in this western blot analysis recognizes only one epitope, which is not present in all isoforms. Other isoforms, therefore, may be expressed in these cell types and not be immunoreactive to the antibody used. B: Park2 and GFAP immunoreactivity in mixed cortical cultures. Primary cultures of cortical neurons and type I astrocytes were double labeled with antibodies against Park2 protein (green signal) and GFAP (red signal), as previously described  ADDIN EN.CITE  ADDIN EN.CITE.DATA [49], and observed by fluorescence microscopy.