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A novel mutation causing nephronophthisis in the Lewis polycystic kidney rat localises to a conserved RCC1 domain in Nek8

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Additional File 1

Table S1: Candidate genes

Genes on chromosome 10 from region 63M to 65.5Mbp, selected as candidate genes for the LPK mutation based on known functional roles.

SYMBOL RGD_ID NAME START STOP Basis for candidate selection

RGD1309077 1309077

Similar to putative RNA

methyltransferase 63600150 63600936

Hypothetical protein associated with RNA binding methyltransferase activity [1].

Phf12 1305731 PHD finger protein 12 64024504 64074603

Transcriptional repressor of activity. Negative regulation of DNA-dependent transcription [1].

Flot2 70993 Flotillin 2 64081463 64104166 Colocalized with activated GPI-linked cell adhesion molecules at the plasma membrane [1].

Spag5 620152

Sperm associated

antigen 5 64313146 64330917

Associated with microtubules, flagella structures and sperm motility [2].

Sdf2 1307274

Stromal cell derived

factor 2 64378702 64389195

Associated with endoplasmic reticulum (stress induced unfolding). Highly expressed in fast growing,

differentiating and meristematic tissues [3].

Supt6h 1309290

Suppressor of Ty 6

homolog (S. cerevisiae) 64388520 64425858 Stimulates transcription elongation [4].

Proca1 1561727

Protein interacting with

cyclin A1 64426828 64440614 Protein interacting with cyclin A1-CDK2 complex [5].

Nek8 1306897

NIMA (never in mitosis

gene a)- related kinase 8 64458887 64470059

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SYMBOL RGD_ID NAME START STOP Basis for candidate selection

Traf4 1306708

Tnf receptor associated

factor4 64469647 64477810

TNF associated receptor protein associated with signal transduction developmental neural crest. Linked to epithelial homeostasis and cell polarity [8, 9].

Sarm1 1310078

Sterile alpha and TIR

motif containing 1 64584101 64607488

Down regulates NF-kappaB and IRF3 mediated TLR3 and TLR4 signalling. May also directly Inhibit MAPK phosphorylation [10].

Ift20 1309400

Intraflagellar transport 20 homolog

(Chlamydomonas) 64647046 64652539

Cilia associated protein linked to PKD cystic kidney (floxed knockout mouse). Increases canonical Wnt signaling. Has basal body & Golgi complex interaction with polycystin 2, kinesin2, Kif3a [11-13].

Nlk 1561602 Nemo like kinase 64681465 64804964

Inhibits canonical Wnt pathway regulation, link to usb4 (ubiquitin pathway) [14].

Ksr1 1308105 Kinase suppressor ras 1 65187915 65322237

A molecular scaffold and positive regulator of the Raf/MEK/ERK phosphorylation cascade [15].

References

1. Rat Genome Database, http://rgd.mcw.edu.

2. Fitzgerald CJ, Oko RJ, van der Hoorn FA: Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers. Mol Reprod Dev 2006, 73(1):92-100.

3. Schott A, Ravaud S, Keller S, Radzimanowski J, Viotti C, Hillmer S, Sinning I, Strahl S: Arabidopsis stromal-derived Factor2 (SDF2) is a crucial target of the unfolded protein response in the endoplasmic reticulum. J Biol Chem 2010, 285(23):18113-18121.

4. Endoh M, Zhu W, Hasegawa J, Watanabe H, Kim DK, Aida M, Inukai N, Narita T, Yamada T, Furuya A et al: Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro. Mol Cell Biol 2004, 24(8):3324-3336.

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iii 6. Otto EA, Trapp ML, Schultheiss UT, Helou J, Quarmby LM, Hildebrandt F: NEK8 mutations affect ciliary and centrosomal localization

and may cause nephronophthisis. J Am Soc Nephrol 2008, 19(3):587-592.

7. Shiba D, Manning DK, Koga H, Beier DR, Yokoyama T: Inv acts as a molecular anchor for Nphp3 and Nek8 in the proximal segment of primary cilia. Cytoskeleton (Hoboken) 2010, 67(2):112-119.

8. Kalkan T, Iwasaki Y, Park CY, Thomsen GH: Tumor necrosis factor-receptor-associated factor-4 is a positive regulator of transforming growth factor-beta signaling that affects neural crest formation. Mol Biol Cell 2009, 20(14):3436-3450.

9. Kedinger V, Alpy F, Baguet A, Polette M, Stoll I, Chenard MP, Tomasetto C, Rio MC: Tumor necrosis factor receptor-associated factor 4 is a dynamic tight junction-related shuttle protein involved in epithelium homeostasis. PLoS One 2008, 3(10):e3518.

10. Peng J, Yuan Q, Lin B, Panneerselvam P, Wang X, Luan XL, Lim SK, Leung BP, Ho B, Ding JL: SARM inhibits both TRIF- and MyD88-mediated AP-1 activation. Eur J Immunol 2010, 40(6):1738-1747.

11. Follit JA, San Agustin JT, Xu F, Jonassen JA, Samtani R, Lo CW, Pazour GJ: The Golgin GMAP210/TRIP11 anchors IFT20 to the Golgi complex. PLoS Genet 2008, 4(12):e1000315.

12. Jurczyk A, Gromley A, Redick S, San Agustin J, Witman G, Pazour GJ, Peters DJ, Doxsey S: Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly. J Cell Biol 2004, 166(5):637-643. 13. Baker SA, Freeman K, Luby-Phelps K, Pazour GJ, Besharse JC: IFT20 links kinesin II with a mammalian intraflagellar transport

complex that is conserved in motile flagella and sensory cilia. J Biol Chem 2003, 278(36):34211-34218.

14. Thorpe CJ, Moon RT: nemo-like kinase is an essential co-activator of Wnt signaling during early zebrafish development. Development

2004, 131(12):2899-2909.

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MEKYERIRVVGRGAFGIVHLCLRKADQKLVIIKQIPVEQMTKDERLAAQNECQVLKLLSHPNVIEYYENFLEDKALMIAMEYAPGGTLAEYIQK

RCNSLLDEDTILHFFVQILLALHHVHTKLILHRDLKTQNILLDKHRMIVKIGDFGISKILSSKSKAYTVVGTPCYISPELCEGKPYNQKSDIWALGCV

LYELASLKRAFEAANLPALVLKIMSGTFAPISDRYSPELRQLILSMLNLDPSKRPQLNEIMAQPICIRPLLNLYTDVGSVKMRRVEKPLAPVPTVP

HGRTGSRISSARSRGVRRGPARPGIPPPLSSVYTWGSGISTPLRLPMLNTEVVQVSAGRTQKAGVTKSGRLIMWEAPPMGTAGGPSLPGAV

EQLQPQFISRFLEGQSGVTIKHVSCGDLFTACLTDRGIIMTFGSGSNGCLGHGNFTDVSQPKIVEALLGYEMVQVSCGASHVLAVSNEREVFA

WGRGDNGRLGLGTQESHNSPQQVTVPPEHEAQRVVCGIDSSMILTVQNQILACGSNRFNKLGLDRISSAEEPSPEDQVEEAHSFTPVQSAP

LNQEPIVCADIGTAHSAAVTASGQCYTFGSNQHGQLGTNARRNSRVPCLVAGLQGMKVTMVACGDAFTVAIGAEGEVYTWGKGARGRLG

RRDEDTGIPKPVQLEETHPYVVTSVACCHGNTLLAVKP5

Ancestral DNA sequence and evolutionary conserved amino acids in the NEK8 protein.

The mutation identified in the present study at R650C is located in one of the repeat motifs within the RCC1 domain as indicated by the red box. Blue text and orange text indicates conserved sequence, grey text indicates non-conserved sequence. The serine/threonine kinase region of the protein (N-terminus) shows evolutionary conservation (blue). The intermediate region of the protein shows non-conserved regions (grey), while the C-terminal shows a mixed pattern (grey and blue). The orange text highlights the G[QRC]LG motif, which was used in subsequent analysis to identify proteins with a related biological function. The R650C lies within one of these domains. The evolutionary history was inferred using the Maximum Likelihood method based on the JTT matrix-based model in MEGA5 [1].

References

1.  Tamura K, Peterson D, Peterson N, Stecher G, Nei M, kumar S: MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011, 28(10):2731-2739.

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RCC1 Repeat From To Fragment

RCC1-Rep_1 317 355 LSSVYAWGGGLSIPLRLPMMPNTEVVQVAAGRTQKAGVTR

Non_Canonical_2 357 414 GRLILWEAPPLGTGGGTLLPGAVELPQPQFVSRFLEGQSGVTIKHVACGDLFTACLTD

RCC1-Rep_3 415 466 RGIIMTFGSGSNGCLGHGSLTDISQPTIVEALLGYEMVQVACGASHVLALSA

RCC1_Rep_4 467 518 DGELFAWGRGDGGRLGLGTRESHNCPQQVPMVPGQEAQRVVCGIDCSMILTS

RCC1_Rep_5 520 571 GRVLACGSNRFNKLGLDCLSLEEEPVPHPQQVEEALSFTPLGSAPLDRETLLC

RCC1_Rep_6 585 636 SGACYTFGSNQHGQLGTSSRRVSRAPCRVQGLEGIKMVMVACGDAFTVAIGA

RCC1_Rep_7 638 689 GEVYSWGKGARGRLGRRDEDAGLPRPVQLDETHPYTVTSVSCCHGNTLLAVR

Additional file 3: Figure S2.

RCC1 domains.

 

Figure illustrates predicted RCC1 domains as defined by the web based program REP (RCC1 repeat, left column) and their alignment around the RCC1 motif (red text), which was used as the basis for three dimensional protein folding predictions. Amino acid positions in the protein are indicated as starting (from) and end (to) positions based on the rat sequence (NP_001099274). Orange bold text highlights the G[QRC]LG motifs, and are also conserved amino acids. An additional RCC1-like repeat domain was annotated as a non-canonical domain as it did not contain the typical repeat motif. Bold green text with box indicates position of the known mutations in Nek8 from human (RCC1-Rep-1,(P), Rcc1-Rep-3 (H) and RCC1-4 (A) ) and mouse (RCC1-Rep-3 (G), jck and the red square highlight the R650C Nek8 LPK mutation (RCC1-Rep-7 (R).

References

1. Bork P, Andrade MA, Ponting CP, Gibson TJ: Homology-based method for identification of protein repeats using statistical significance estimates. J Mol Biol 2000, 298(3):521-537. 2. Otto EA, Trapp ML, Schultheiss UT, Helou J, Quarmby LM, Hildebrandt F: NEK8 mutations affect ciliary and centrosomal localization and may cause nephronophthisis. J Am Soc Nephrol 2008, 19(3):587-592.

3. Liu S, Lu W, Obara T, Kuida S, Lehoczky J, Dewar K, Drummond IA, Beier DR: A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.

Development 2002, 129(24):5839-5846.

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C  

1000  μm  

C  

 

 

 

D  

A  

 

 

 B  

Additional file 4: Figure S3.

Determination of cyst to kidney ratio.

Kidney sections from a heterozygote BC1 animal (A) and homozygote BC1 animal (C) stained with H&E. Panels B and D show the same respective sections after contrast enhancement to allow determination of cyst area (black) relative to total kidney area (white) within the defined kidney region. The data was then subjected to factor analysis alongside other phenotypic variables using principal components to identify traits with the most predictive power for LPK. Scale bar in panel D is 1000µm for all panels.

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Additional File 5

Table S2: Primer List and genomic location based on rn4 rat

assembly

Primer BC START bp Primer F2 START bp

D10Rat43 23,428,128 D10Rat43 23,428,128

D10Rat38 31668581

D10Rat173 37,505,290

D10Rat104 41,968,132

D10Rat83 46860048

D10Rat77 56897760

D10Rat160 59,053,888 D10Rat160 59053888

D10Rat80 61,798,249 D10Rat80 61,798,249

D10Rat159 63166992

D10Rat30 63,752,513 D10Rat30 63,752,513

D10Mit2 64860950

D10Mco16 65,350,890

D10Mgh14 65,379,851

D10Got82 65,996,480

D10Rat161 67091545

D10Rat242 67141196

D10Mgh6 67,677,924

D10Arb27 69,160,380

D10Rat28 69,969,510

D10Rat155 70,229,782

D10Rat58 71,067,496 D10Rat58 71,067,496

D10Rat26 72,105,962 D10Rat26 72,105,962

D10Rat220 72,285,504 D10Rat220 72,285,504

D10Rat57 74,388,383

D10Rat86 82,474,813

D10Rat17 95,066,310

D10Rat12 99,198,839

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ii Table S2

Figure

Table S1: Candidate genes
Table S2: Primer List and genomic location based on rn4 rat assembly

References

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