Sam68 Regulates a Set of Alternatively Spliced Exons during Neurogenesis

Abstract

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.Alternative splicing allows multiple functionally distinct mRNAs and proteins to be generated from a single gene, greatly enhancing the coding potential of the genome. Splicing patterns are controlled by RNA binding proteins that recognize specific splicing enhancer and silencer elements in the premRNA to alter spliceosome assembly, and variation in the expression of these proteins leads to tissue-specific exon use (5, 38). Splicing within a single cell can also be dynamically controlled by extracellular stimuli, although how this information is transmitted to splicing regulatory proteins is not yet known (55).Sam68 is a nuclear RNA binding protein implicated in various aspects of mRNA metabolism, including splicing, nuclear export, somatodendritic transport, and translation. Sam68 belongs to the family of GSG (GRP33, Sam68, GLD1) or STAR (signal transduction and activation of RNA) domain proteins. This domain includes a central KH (hnRNP K homology) RNA binding domain flanked by conserved N and C termini (36,54,59). The GSG domain in Sam68 binds to RNA motifs that are rich in A or U, such as UAAA or UUUA, and also mediates homodimerization (9, 31). Sam68 contains a variety of other protein domains that allow its interaction and modification by multiple signaling pathways. These many regulatory interactions and posttranslational modifications affect the RNA binding activity and localization of Sam68 and make it an appealing molecule for transducing information from signaling systems to pathways of mRNA metabolism (13,33,46,51,60).Sam68 is localized primarily in the nucleus as observed by immunofluorescence, consistent with its role in alternative splicing. Sam68 helps regulate the splicing of CD44 variable exon v5 in response to phosphorylation by extracellular signalregulated kinase in T lymphoma cells (39). Sam68 binds to exonic regulatory elements of v5 and cooperates wi...

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells

“…[14][15][16] An example of RBP that is upregulated in several human tumors is SAM68 (Src-associated in mitosis of 68 kDa), a member of the STAR (Signal Transduction Activator of RNA metabolism) family of RBPs. 17 SAM68 is involved in regulating several aspects of RNA processing, such as transcription, [18][19][20][21] AS [22][23][24][25] and translation of cellular mRNAs. [26][27][28] Moreover, SAM68 displays pro-oncogenic functions and is frequently upregulated in human cancer types, 17 including prostate cancer (PCa), wherein it supports cell proliferation and survival to genotoxic stresses.…”

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

“…Deletion of this element promoted the skipping of Rps6kb1 exons 6a, 6b, and 6c, thus preventing the expression of Rps6kb1-002. Sam68 is an established regulator of alternative splicing, and it is known to function by directly associating with A/U-rich elements near 5= splice sites (5,10,12,16). The Rps6kb1 intron 6 UAAU UAAA bipartite sequence is recognized by Sam68 with relatively high affinity.…”

“…Sam68 regulates the epithelial-to-mesenchymal transition by decreasing the presence of an alternative serine/arginine-rich splicing factor 1 gene (Srsf1) transcript degraded by nonsense-mediated mRNA decay (9). Sam68 has been shown to regulate alternative splicing of mRNAs during neurogenesis (10) and in cerebellar neurons (11). Stimulation of cerebellar neurons using the glutamate receptor agonist kainic acid was dramatically attenuated without Sam68, indicating that Sam68 is required for activity-dependent alternative splicing of Nrxn1 in vivo (11).…”

Emerging roles for Sam68 in adipogenesis and neuronal development

“…12 In addition, it was demonstrated that Sam68 was required for maintaining patterns of splicing even after differentiation had occurred. As Sam68 knockout mice exhibit defects in basal motor coordination, 4 the alterations in splicing patterns may partly contribute to this defect.…”