Alternative splicing accounts for the four forms of myelin basic protein

Proteomic diversity is frequently achieved by alternative RNAsplicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system. quaking (qk), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor. The qk locus encodes several RNA-binding proteins with heterogeneous nuclear ribonucleoprotein K-type homology, a characteristic of several known alternative splicing regulators. Here we test the nuclear-localized qk isoform (QKI-5) for its ability to regulate alternative splicing of MAG pre-mRNA in transient coexpression assays. QKI-5 exhibits properties of a negative regulator of MAG exon 12 alternative splicing. An intronic sequence element required for the repressive function and binding of QKI-5 is also identified. Direct evidence for irregularities in alternative splicing of MAG and other myelin protein transcripts in the qk mouse is demonstrated.A lternative splicing is a powerful way to regulate gene expression at the posttranscriptional level and to generate protein diversity (1, 2). Understanding the molecular basis of such mechanisms is an urgent challenge, because the number of protein-coding genes in humans has been estimated at barely double the number in Drosophila and Caenorhabditis elegans. Between 35% and 59% of human genes are predicted to generate alternatively spliced mRNA isoforms (3).Mouse quaking (qk), also known as quaking viable (qk v ), is a classical recessive dysmyelination mutation (4). The qk locus produces a diverse set of proteins by alternative splicing (5, 6). They all contain a single heterogeneous nuclear ribonucleoprotein K homology (KH) RNA-binding domain and belong to the evolutionarily conserved signal transduction and activator of RNA (STAR) family (7,8). The first three studied in detail are constructed with the same 311-aa body, but have different carboxyl tails. QKI-5 is the only nuclear isoform and shuttles between the nucleus and cytoplasm (9, 10). The expression of QKI isoforms is developmentally regulated, with QKI-5 being highly expressed throughout the embryogenesis and neonatal stages and decreasing gradually thereafter (7, 9). In postnatal day 14 (P14) mutant mice QKI proteins are decreased exclusively in myelin-forming cells. In addition, the QKI-5 expression level in qk v brain correlates with the severity of dysmyelinating phenotype, suggesting a function of QKI-5 in myelination (9).The relationship between the decreased QKI protein in affected mice and their myelination defects is not understood. It has been shown that several myelin-specific genes are alternatively spliced, including myelin basic protein (MBP), proteolipid protein (PLP), and MAG (11-13). Some splicing events seem to be abnormal in qk v mice. The best-documented c...

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“…Notably, several other myelin genes are also alternatively spliced (12,13). Among them, PLP and MBP represent possible targets of QKI-5.…”

Section: Resultsmentioning

confidence: 99%

“…It has been shown that several myelin-specific genes are alternatively spliced, including myelin basic protein (MBP), proteolipid protein (PLP), and MAG (11)(12)(13). Some splicing events seem to be abnormal in qk v mice.…”

mentioning

confidence: 99%

Function of quaking in myelination: Regulation of alternative splicing

Reed

Grabowski

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

182

214

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“…1B). Their expression is enriched during active early myelination in the human and mouse, and in immature OLGs in culture [5][6][7]. These minor MBP isoforms have been observed in the nucleus of primary OLG cultures and OLGs in the mouse brain [25][26][27] and are also enriched in the radial component of compact myelin [28,29].…”

Section: Introductionmentioning

confidence: 97%

“…One of the major families of proteins of CNS myelin arises from the Golli (Gene of Oligodendrocyte Lineage) complex, which is responsible for the developmentally-regulated production of early developmental Golli proteins, and classic myelin basic proteins (MBPs) [3][4][5]. The classic MBP isoforms arise from transcription start site 3 of Golli and are nominally 14-kDa, 17.22/17.24-kDa, 18.5-kDa, and 21.5-kDa in mammals [6,7], and are essential for the formation of compact CNS myelin by OLGs [8,9]. The 18.5-kDa isoform predominates in adult human myelin and has been the most studied.…”

Section: Introductionmentioning

confidence: 99%

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

Smith

Seymour

Boggs

et al. 2012

Biochemical and Biophysical Research Communications

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The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca 2+ -calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two nontraditional PYnuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

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“…MBP exists as several different isoforms that result from alternative splicing of the primary mRNA transcripts (de Ferra et al, 1985;Kamholz et al, 1986Kamholz et al, , 1987Kamholz et al, , 1988Streicher and Stoffel, 1989;Asipu and Blair, 1997;Givogri et al, 2000), and multiple isoforms have been cloned and identified from human and mouse . In fish, several MBP cDNAs were reported (Spivack et al, 1993;BrÖ samle and Halpern, 2002).…”

Section: Introductionmentioning

confidence: 99%