Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies
Authors: Paul De Koninck
Edouard W Khandjian
Sandra Tremblay
Alain Y Dury
Rachid El Fatimy
Timothy M Rose
Jocelyn Côté
Journal: PLoS Genetics (2013)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Fragile X syndrome; FMRP; RNA-binding protein; neurons; mRNA transport
Affiliations: Centre de recherche, Institut en santé mentale de Québec, Québec, Québec, Canada ; Département de psychiatrie et des neurosciences, Faculté de médecine, Université Laval, Québec, Québec, Canada.

Abstract:  Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome. 

Retracted: No

PubPeer comments:
 It is known that GFP generates nuclear aggregates when fused with a NLS. For this reason it is not clear why the authors claim that GFP-nuclear isoforms of FMRP (namely ISO6 and ISO12) are colocalized with Cajal bodies without: -ttesting the localization of ISO6 and or ISO12 fused to a small tag (e.g. Flag) -ttesting the nuclear localization of transfected ISO12 and ISO6 (not fused to any tag) by using an anti-FMRP antibody, such as, for instance, 1C3 recognizing the N-terminal portion of FMRP. Indeed, other authors followed this strategy and showed that ISO12 is localized in the perinucleolar regions (Ramos et al. Structure, 2006). This strongly suggests that the colocalization of (GFP)-FMRP with Cajal bodies is likely artifactual. The authors claim that that their home-made antibody is specific for FMRP and recognizes the nuclear FMRP colocalized with coilin. However the key experiment to prove that claim is not shown. , 
 Response to Mrs Mr Halictus Langobardicus 1)*** It is known that GFP generates nuclear aggregates when fused with a NLS. *** See our section Material and Methods, paragraph Nucleic acids studies, line 14 from bottom. 2)* **Testing the nuclear localisation of transfected ISO12 and ISO6 by using an anti-FMRP antibody, such as for instance 1C3.*** This has been done years ago : see Sittler et al. HMG 1999 and Davidovic et al. HMG 2005. 3)* **Other authorsu2026showed that ISO12 is localized in the perinucleolar regions (Ramos et al. Structure, 2006).** * CB are dynamic entities that can be found at the nucleolar periphery (see Gall 2000, Annu. Rev. Cell Dev. Biol 16 ; Platini et al. 2000, JCB). Note that Cajal himself observed the u00ab accessory body u00bb close to or touching the nucleolus, more than a century ago. 4)* **Concerns about the antibody IgY#C10*** Obviously, the author is not familiar with FMRP. IgY#C10 has been succesfully used in : El Fatimy et al. 2012, PlosOne ; Dury et al. 2013, PlosGenet ; Moutaoufik et al. 2014, PlosOne ; El Fatimy et al. 2016, PlosGent ; Leboucher et al. 2019, Mol Metab. Concerning mAb1C3 (previously referred to as mAb1a), it does not work in IP (Ceman et al. 1999, MCB ; see also data sheets from suppliers), and slightly cross reacts with FXR1P in WB analyses (Khandjian et al. 1998, HMG ; and data sheets from suppliers). , 
 Do ISO6 or ISO12 fused to Flag His HA-tag have the same localization as GFP-ISO6 or ISO12 or the same localization as the untagged proteins? tested ? If not, the risk is to support an artifact CB are dynamic but that is not what the authors are showing here: the experiments were performed in fixed cells and in contradiction with the localization of the untagged proteins (Ramos et al., 2006). El Fatimy co-workers do not show any perinucleolar distribution of GFP-ISO6 ISO12. The first conclusion is that the different localization between the two works is caused by the presence of the GFP tag. 3 papers describing CLIP results have been published using different anti-FMRP antibodies. (Darnell, Cell 2011; Tabets, PNAS, 2016 ; Maurin, NAR, 2018. Overlapping results are shown between Cell NAR papers). This means that anti-FMRP antibodies able to immunoprecipitate this protein exist and could be used. The authors can claim what they want but simple controls need to be performed. ,