Conclusion-3 RGG-domain of Top3? require for its participation

Conclusion-3·        SNVof Top3?from autism with C666R mutation  lost orreduced its RNA topoisomerase activity whereas other SNV with R472Q mutationfrom schizophrenic patient retain its RNA topoisomerase activity comparing tothe wild typed  Top3?·        Top3?-C666R lost 10 folds of mRNA bindingcapacity while Top3?-R472Qretain mRNA binding capacity comparing to Top3?-WT·        Association with FMRP and Top3?-C666R and Top3?-R472Q is impaired but with TDRD3they formed complex with 30% less amount comparing with Top3?-WT.·        Top3?-C666R variant lacked its ability torescue abnormal synaptic branches and boutons numbers at NKJs of Drosophila  DiscussionTop3?is the major mRNA binding topoisomerase in human cells. Top1, Top2?, Top2?, Top3? and Top3? are 5 topoisomerases of human cells.

Among two Type-IA topoisomerase only Top3? captured as major mRNA binding topoisomerase by mRNAbinding capture assay and immunoblotting. The relative ratio of Top3?, TDRD3 and FMRP for mRNA bindingcapacity is comparable with each other while not comparable with positivecontrol TIA1. This observation supports the notion that individual binding of Top3?, TDRD3 and FMRP is not that muchstrong and requires the association with each other to interact strongly withmRNA.Both catalytic Tyr residue and RGG-domain of Top3? require for its participation innormal synapse formationDeletion of the RGG-box leads toimpair binding with mRNA along with the impairment in rescue of abnormalsynaptic branches and boutons numbers at NMJs of Drosophila. Although Top3?-Y336F mutant lost their RNAtopoisomerase activity but retain its mRNA binding capacity.

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But the fact is, Top3?-Y336F mutant also failed to promotenormal synapse formation.SNVs of Top3?from schizophrenic and autism patient have impaired in vivo function Top3?-R472Q & Top3?-C666R both variant failed toassociate with FMRP. FMRP is an important RBP and mutation of FMRP causesFrazile X syndrome (leading cause of autism). Both the variants retainassociation with TDRD3 but this association seems not sufficient to rescueabnormal synaptic branches and boutons numbers in Drosophila with Top3?-C666R mutant. In Top3?-R472Q mutant positively charged R replaced by non-charged Qand non-charged C replaced by positively charged R in Top3?-C666R which explain theirimpairment to mRNA targeting, RNA topoisomerase activity and other in vivo functions.Significance•      Top3? acts not only asRNA topoisomerase but also regulates the mRNA translation by associating withpolyribosome and this function conserved in human to chicken to fruit flies•      Deletionof catalytic Tyr residue leads to deficient of RNA topoisomerase activity butthe normal mRNA binding activity remains but failed to promote synapse formation•      RGG-domaindeletion also leads to decreased targeting of Top 3? to mRNA and also failed to promoteneurodevelopment•      Oneaspect is needed to be mentioned that as in vivo RNA topoisomerase activity ofTop 3? is not evident,but for its DNA topoisomerase activity also requires both the catalytic Tyrresidue and the unique RGG-box in its sequence.

So Whether Top3? a RNA or DNA topoisomerase it isimportant for neurodevelopment.Comments/Conclusion·        Invivoevidence of RNA topoisomerase activity of Top3? will substantiate the research findings·        Between Top3?and TDRD3 which one is more crucial not clear by the study? As Top3? can associate with mRNA through atleast three ways- through RGG-domain of Top3?, through TDRD3 itself or throughthe EBM domain or the TUDOR domain of TDRD3.  So the light band of ?RGG mutant after longexposure (Figure-5), can be explained by this way but not clears fully.·        Why only Top3?-C666Rmutant impaired in RNA topoisomerase activity and mRNA binding capacity? Whynot Top3?-R472Qvariant? This question needs further investigations.·        Roleof Nuclear localization sequence (NLS) in WT-Top3? to bind with mRNA and to promotesynapse formation needs extensive investigation.

·        Identification of genes regulated by Top 3?-TDRD3 complex may provide targets for therapeuticintervention·        Whether Top1 and Top2? are RNA topoisomerase or not?14,15Overallcurrent study is unique and significant enough to publish in the journal andwill add a positive wing into this field of research.References1.Wang,J.C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective.Nat.

Rev. Mol. Cell Biol., 3, 430–440.2.Pommier,Y., Sun,Y., Huang,S.

N. and Nitiss,J.L. (2016) Roles of eukaryotictopoisomerases in transcription, replication and genomic stability. Nat.Rev.

Mol. Cell Biol., 17, 703–721.3.Forterre,P., Gribaldo,S., Gadelle,D.

and Serre,M.C. (2007) Origin and evolutionof DNA topoisomerases. Biochimie, 89, 427–446.4.Xu,D.,Shen,W.

, Guo,R., Xue,Y., Peng,W.

, Sima,J., Yang,J., Sharov,A.

, Srikantan,S.,Fox,D. 3rd et al. (2013) Top3beta is an RNA topoisomerase that workswith fragile X syndrome protein to promote synapse formation. Nat. Neurosci.,16, 1238–1247.5.

Wang,H., Di Gate,R.J.

and Seeman,N.C. (1996) An RNA topoisomerase. Proc.Natl. Acad. Sci.

U.S.A., 93, 9477–9482.6.Siaw,G.E., Liu,I.

F., Lin,P.Y., Been,M.D. and Hsieh,T.

S. (2016) DNA and RNAtopoisomerase activities of Top3beta are promoted by mediator protein Tudordomain-containing protein 3. Proc. Natl.

Acad.Sci. U.S.A., 113, E5544–5551.

7.Ahmad,M., Xue,Y., Lee,S.

K., Martindale,J.L., Shen,W., Li,W.

, Zou,S.,Ciaramella,M., Debat,H., Nadal,M. et al. (2016) RNAtopoisomerase isprevalent in all domains of life and associates with polyribosomes in animals.

NucleicAcids Res., 44, 6335–6349.8.Yang,Y.,McBride,K.M., Hensley,S., Lu,Y.

, Chedin,F. and Bedford,M.T. (2014) Argininemethylation facilitates the recruitment of TOP3B to chromatin to prevent R loopaccumulation.

Mol. Cell, 53, 484–497.9.Darnell,J.C., Van Driesche,S.J.

, Zhang,C., Hung,K.Y., Mele,A., Fraser,C.E.,Stone,E.F.

, Chen,C., Fak,J.J., Chi,S.

W. et al. (2011) FMRP stallsribosomal translocation on mRNAs linked to synaptic function and autism. Cell,146, 247–261.

10.Iossifov,I., Ronemus,M., Levy,D., Wang,Z., Hakker,I., Rosenbaum,J.

, Yamrom,B.,Lee,Y.H., Narzisi,G., Leotta,A. et al. (2012) De novo gene disruptionsin children on the autistic spectrum. Neuron, 74, 285–299.

11.Xu,B., Ionita-Laza,I., Roos,J.

L., Boone,B., Woodrick,S., Sun,Y.

, Levy,S.,Gogos,J.A. and Karayiorgou,M. (2012) De novo gene mutations highlight patternsof genetic and neural complexity in schizophrenia. Nat. Genet., 44,1365–1369.

12.Castello,A., Horos,R., Strein,C.

, Fischer,B., Eichelbaum,K., Steinmetz,L.M.,Krijgsveld,J. and Hentze,M.W. (2013) System-wide identification of RNA-bindingproteins by interactome capture.

Nat. Protoc., 8, 491–500.13.Bauer,W.

J., Heath,J., Jenkins,J.L. and Kielkopf,C.

L. (2012) Three RNArecognition motifs participate in RNA recognition and structural organizationby the pro-apoptotic factor TIA-1. J.

Mol. Biol., 415, 727–740.14.

Castello,A., Fischer,B., Eichelbaum,K., Horos,R., Beckmann,B.M.,Strein,C.,Davey,N.

E., Humphreys,D.T., Preiss,T., Steinmetz,L.M.

et al. (2012)Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell,149, 1393–1406.15.

Baltz,A.G., Munschauer,M.

, Schwanhausser,B., Vasile,A., Murakawa,Y.,Schueler,M., Youngs,N., Penfold-Brown,D., Drew,K.

, Milek,M. et al. (2012)The mRNA-bound proteome and its global occupancy profile on protein-codingtranscripts. Mol.

Cell, 46, 674–690.