Dataset Information

Dynamic role of Rotatin in mitosis, ciliogenesis and nucleokinesis correlates with variable mutant cerebral phenotypes.

ABSTRACT: Recessive mutations in RTTN, encoding centrosome associated protein Rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time a wide variety of other brain malformations has been ascribed to RTTN mutations, such as primary microcephaly. However, the function of Rotatin is largely unknown and the molecular disease mechanism underlying these malformations has not yet been elucidated. Here, we report four novel families identified by high throughput sequencing, one of them with a novel deep intronic variant. We reviewed RTTN-related disease phenotypes and defined the spectrum which includes intellectual disability (ID), short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. Rotatin expression, function and cellular disease phenotype were studied in cultured primary skin fibroblasts from eight unrelated affected individuals. We found that Rotatin deficiency leads to abnormal centrosome amplification during mitosis, resulting in multipolar spindle formation, mitotic failure, increased apoptosis and aneuploidy. In non-dividing healthy cells, Rotatin localizes at the primary cilium, both at basal body and axoneme during ciliogenesis. Interestingly, interphase mutant cells show aberrant ciliogenesis. Proteomics analysis reveals that exogenous Rotatin in HEK293 cells interacts with the non-muscle cellular myosin complex (MYH9-MYH10-MYH14), which is essential for nucleokinesis during neuronal migration. In hiPSC-derived neurons, endogenous Rotatin localizes to the leading edge centrosome, which pulls the perinuclear cage during nucleokinesis, suggesting a pivotal role of Rotatin in neuronal migration. This study sheds light on the pleiotropic cellular functions of Rotatin, explaining disease spectrum linked to both neuronal proliferation and migration.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: Jeroen Demmers

LAB HEAD: Jeroen Demmers

PROVIDER: PXD012415 | Pride | 2019-03-25

REPOSITORIES: Pride

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Publications

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

Vandervore Laura V LV Schot Rachel R Kasteleijn Esmee E Oegema Renske R Stouffs Katrien K Gheldof Alexander A Grochowska Martyna M MM van der Sterre Marianne L T MLT van Unen Leontine M A LMA Wilke Martina M Elfferich Peter P van der Spek Peter J PJ Heijsman Daphne D Grandone Anna A Demmers Jeroen A A JAA Dekkers Dick H W DHW Slotman Johan A JA Kremers Gert-Jan GJ Schaaf Gerben J GJ Masius Roy G RG van Essen Anton J AJ Rump Patrick P van Haeringen Arie A Peeters Els E Altunoglu Umut U Kalayci Tugba T Poot Raymond A RA Dobyns William B WB Bahi-Buisson Nadia N Verheijen Frans W FW Jansen Anna C AC Mancini Grazia M S GMS

Brain : a journal of neurology 20190401 4

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations ...[more]

PMID: 30879067

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Project description:Cellular mRNAs are permanently associated to proteins in the form of ribonucleoprotein particles. In addition to the hnRNP and SR protein families, the double-stranded RNA-binding (DRB) proteins play important roles in mRNA synthesis, modification, activity and decay (Dreyfuss et al, 2002; Stutz & Izaurralde, 2003). Staufen is a DRB protein first described as a maternal factor involved in the localised translation of specific mRNAs during early development in the fly (Riechmann & Ephrussi, 2001). One of the human homologues, human Staufen1 (hStau1) forms ribonucleoprotein complexes known as RNA granules that contain proteins involved in translation regulation as well as cytoskeleton and motor proteins to allow the movement of the granule on the microtubules. Although many cellular mRNAs have been found associated to these RNA granules, the specificity of such binding and the mechanims of hStau1-RNA interaction are still unclear. Here we identified a protected sequence signature with high homology to human Alu family of short interspersed elements at the 3’-untranslated region of mRNAs specifically associated to hStau1 protein. Using a combination of affinity chromatography, RNAse-protection, deep-sequencing and bioinformatic analyses to compare the mRNAs differentially associated to hStau1 or a mutant protein unable to bind RNA we defined a collection of mRNAs specifically associated to wt hStau1. A common sequence signature consisting of two opposite-polarity Alu motifs was present in the hStau1-associated mRNAs and was shown to be sufficient for binding to hStau1 and hStau1-dependent stimulation of protein expression. Our results unravel how hStau1 identifies a wide spectrum of cellular target mRNAs to control their localisation, expression and fate. We suggest that mammalian Staufen1 protein has adapted to use the evolutionary recent Alu elements as recognition signals thereby increasing its possibilities for rapid identification to new mRNA targets. Staufen (wild type or mutant) associated RNAs were extracted from HEK293T cells and sequenced according the illumina mRNA-seq potocol (HiSeq2000 sequencer). A third sample, of RNA, associated to a wild type Staufen, was treated with RNase T1 so only RNA fragments directly attached to Staufen were purified and sequenced.

Dataset Information

Dynamic role of Rotatin in mitosis, ciliogenesis and nucleokinesis correlates with variable mutant cerebral phenotypes.

Publications

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

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