SWITCH TO THE L ISOFORM OF THE MAP/MICROTUBULE AFFINITY-REGULATING KINASE 4 (MARK4) GENE, MAINLY EXPRESSED IN HUMAN GLIOMA, VIA PRE-MRNA ALTERNATIVE SPLICING MODULATED BY POLYPYRIMIDINE TRACT-BINDING PROTEIN (PTB)

MARK4 (MAP/Microtubule Affinity-Regulating Kinase 4) is a serine-threonine kinase that phosphorylates the Microtubule Associated Proteins (MAPs) taking part in the regulation of microtubule dynamics involved in cell cycle regulation and proliferation. MARK4 is ubiquitously expressed and two different isoforms are produced through alternative splicing: the constitutive MARK4S, consisting of 18 exons, and the alternative MARK4L encoded by a transcript that undergoes skipping of exon 16 and consequent frameshift of the reading frame. The differential expression of the two MARK4 isoforms in human tissues and, particularly in the Central Nervous System, point to their not fully overlapping roles. MARK4S is predominant in normal brain and differentiated neurons and has been associated to cell differentiation, while MARK4L is mainly expressed in neural progenitors and is up-regulated in hepatocarcinoma and glioma, suggesting a function of this isoform in cell proliferation. In glioma and glioblastoma-derived cancer stem cells (GBM CSCs), we pointed out an imbalance between the two MARK4 isoforms, directly proportional to cell de-differentiation and tumour grade. Such imbalance is triggered by decrease in MARK4S expression, associated with overexpression of MARK4L. The MARK4 expression profile observed in glioma matches that observed in mouse neural stem cells (NSCs). Moreover, MARK4L has been found expressed in the embryonic ventricular zone and adult sub-ventricular zone, both well known regions of neurogenesis, suggesting that the balance between the two MARK4 isoforms is critical for neural differentiation and proliferation. Therefore, in glioma a subverted MARK4L/MARK4S ratio may contribute to cellular de-differentiation and proliferation during gliomagenesis. Having ruled out mutations or copy number loss/gain as cause of deregulation of MARK4 expression in glioma, we hypothesised that alterations at the post-transcriptional level, possibly in alternative splicing, might be at the origin of the observed MARK4 isoforms imbalance. MARK4L total mRNA evaluation by real-time PCR in glioma tissue samples and GBM CSCs, failed to reveal differences in the overall expression of the kinase, even in the presence of significantly different expression levels of the two isoforms, supporting the hypothesis of an alteration in alternative splicing at the root of MARKL/MARK4S imbalance in glioma. Alternative splicing is the most important mechanism in generating proteomic diversity from a limited number of genes. Alternatively spliced isoforms expression is thinly controlled according to specific cells signalling and developmental stages by a complex interplay between spliceosome and splicing regulatory factors, which can be grouped into the two main classes of SR (serine-arginine rich) and hnRNP (heterogenous nuclear ribonucleoprotein) proteins. The intrinsic plasticity of alternative splicing in proteome modelling, make sthis process a tempting target for cancer cells to enhance the production of protein isoforms sustaining tumour growth and spread. Protein isoforms produced from exon skipping, like MARK4L, are commonly found enriched in cancer, including glioma, as a consequence of hnRNP protein overexpression. We thus performed bioinformatic analysis to identify putative binding sites for hnRNP proteins in MARK4 pre-mRNA. Among the predicted putative binding sites for hnRNPs, three binding motifs for PTB (polypyrimidine-tract binding protein) appeared of particular interest, given the role of PTB in regulating neural stem cells proliferation and differentiation and its established involvement in glioma, where it is aberrantly overexpressed and induces the expression of protein isoforms produced by exon skipping. We found two putative PTB binding sites in intron 15 (IVS15) and one in intron 16 (IVS16). One of the two identified regulatory sequences in IVS15 is embedded in a polypyrimidine rich context and may represent a high affinity PTB binding site that could favour MARK4 exon 16 skipping by competition with U2AF65 or, together with the other PTB binding sites, by polymerisation or intron looping. A functional role of these sites is also suggested by the high sequence conservation between human and mouse. Western blot analysis showed a significant overexpression of PTB in our astrocytoma and glioblastoma samples, correlating with MARK4L expression and thus with tumour grade and cell de-differentiation. Moreover, GBM CSCs, both undifferentiated and differentiated, showed high levels of PTB coherently with the nearly exclusive expression of MARK4L. However, while NSCs show undetectable levels of MARK4L upon differentiation, in differentiated GBM CSCs high expression levels of MARK4L are maintained possibly sustained by PTB overexpression. We then performed minigene splicing assays to identify the specific sequences involved in MARK4 alternative splicing. We focused our attention on the polypyrimidine tract in MARK4 VS15 and constructed a MARK4 splicing minigene by cloning, in a mammalian expression vector, the MARK4 genomic region comprising exons 15, 16 and 17 and IVS15 and IVS16. Sequential deletions of different IVS15 portions revealed that the last 87 nucleotides of intron 15 contain a functional intronic splicing silencer (ISS). However, mutagenesis of the PTB binding site contained in this region did not affect minigene splicing, suggesting that PTB may be involved in MARK4 splicing by binding to a non canonical ISS or by cooperating with other identified PTB binding sites. Moreover, electrophoretic mobility shift assays (EMSA) highlighted a specific shifted band probably due to the interaction of splicing factors with MARK4 pre-mRNA. Mass spectrometry experiments are in progress aiming at identifying the protein(s) bound to MARK4 IVS15, and seem to confirm the presence of PTB in the shifted band. The achieved data, suggest that PTB overexpression in glioma may favour MARK4L expression causing the observed imbalance between the two MARK4 isoforms, featuring alternative splicing as an oncogenic mechanism that through the fine tuned regulation of MARK4 isoforms may foster proliferation and de-differentiation in glioma.