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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01226

Journal of Cell Science 117, 3691-3702 (2004)
Published by The Company of Biologists 2004
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KIN17 encodes an RNA-binding protein and is expressed during mouse spermatogenesis

Ghislaine Pinon-Lataillade1,*, Christel Masson1,{ddagger}, Jacqueline Bernardino-Sgherri2, Véronique Henriot1,§, Philippe Mauffrey1, Yveline Frobert3, Silvia Araneda4 and Jaime F. Angulo1,*

1 Laboratoire de Génétique de la Radiosensibilité, CEA, Direction des Sciences du Vivant (DSV), Département de Radiobiologie et de Radiopathologie (DRR), B.P. 6, 92265 Fontenay aux Roses CEDEX, France
2 Laboratoire de la Radiosensibilité des Cellules Germinales, CEA, DSV, DRR, U 566 CEA-INSERM-Université Paris VII, B.P. 6, 92265 Fontenay aux Roses CEDEX, France
3 Service de Pharmacologie et d'Immunologie, Département de Recherche Médicale, DSV, CEA-Saclay, 91191 Gif-sur Yvette, France
4 Physiologie Integrative, Cellulaire et Moléculaire, UMR 5123 CNRS/UCB Lyon 1, 43 Bd. 11 novembre 1918, 69622 Villeurbanne CEDEX, France



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  		Fig. 1. Expression of Kin17 mRNA and protein in developmental and adult mouse testes. (A) Total RNA from testes of mice at days 5, 12, 17, 22, 28, 48 and 53 p.p. were subjected to RT-PCR. PCR products were analysed in a 2% agarose gel. The results shown are from two independent experiments done in triplicate. The numerical interpretation of a representative gel is shown. ß Actin RNA was determined as reference. The intensity of the bands was quantified using NIH image software. The ratios Kin17:ß Actin RNA were calculated and plotted. (B,C) Western analysis of KIN17, PCNA and ß ACTIN proteins from extracts of mouse testes. (B) 20 µg of the detergent-extractable protein fraction and (C) 20 µg of the protein remaining in the pellet were loaded per lane. The positive control (PC) includes purified mouse KIN17 protein produced in bacteria.

 


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  		Fig. 2. Immunohistochemical detection of KIN17 (panels A,B,E,F) and PCNA (panel D) proteins during development of mouse testis. Nuclear staining of Sertoli cells (Ser) for KIN17 was observed in testis sections of 1-day-old (panel A), 5-day-old (panel B), 17-day-old (panel E) and 28-day-old (panel F) mice. Nuclei of spermatogonia (Sg), leptotene (S), pachytene (P) and round spermatid (Rs) cells also labelled for KIN17 (panels B,E,F). No staining was detected in gonocytes (unlabelled bold arrows, panel A). Note the nuclear staining of spermatogonia and leptotene spermatocytes for PCNA in 17-day-old testis whereas pachytene and Sertoli cells are unstained (panel D). A control section of a 5-day-old mouse testis is also shown (panel C). Bars, panels A, B and C: 12 µm, panel D: 20 µm, panel E: 18 µm, panel F: 15 µm. In adult mouse testis, staining for KIN17 protein was observed in the nuclei of Sertoli cells (Ser), spermatogonia (Sg) and on chromosomes of cells undergoing mitosis (mi); spermatocyte cells: leptotene (S) zygotene (Z), pachytene (P), early round spermatid (Rs), in the cytoplasm of elongating and late spermatids (Es) and in residual bodies (Rb) (panels G-K). Arrowheads in panel H show nonidentified Es cytoplasmic spherical components. A control testicular section where anti-KIN17 antibody was replaced by normal mouse IgG remained unstained (panel L). Stages of the seminiferous epithelium are indicated with Roman numerals. Bars: panels G, H and J, 12 µm; panel I, 19 µm; panel K, 10 µm and panel L, 11 µm.

 


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  		Fig. 5. Detection of KIN17 protein in adult mouse testis after nuclease digestion. Immunohistochemical detection of KIN17 protein (D,G,J,M) in testicular sections digested either with DNase I (E,F,G), or with RNase A (H,I,J), or in a combination of the two (K,L,M). DAPI staining (A, control) shows that DNase I digestion released DNA from nuclei of testicular cells, except elongated spermatid heads (E) and that DNA is preserved by RNase A digestion (H). PI staining shows that DNA and RNA are released from nuclei by DNase and RNase digestion (K). Whatever the treatment, a residual staining for KIN17 is observed in cell nuclei; cytoplasmic staining of elongating spermatids is well preserved (arrowheads in D, G, J and M) compared to control (D). SC35 detection is not affected by DNase I digestion (F) compared to control (B) and RAD 51 is not affected by RNase digestion (I) compared to control (C). A negative control where anti-KIN17 mAb was preadsorbed with pure KIN17 protein is shown (L). Ser, Sertoli cells; S, preleptotene, leptotene; Z, zygotene; P, pachytene spermatocytes; Rs, round spermatides; Es, elongated spermatids. Bars: A,E,H, 80 µm; B,C,D,F,G,I, 22 µm; J,L,M, 23 µm; K, 55 µm.

 


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  		Fig. 3. In vivo colocalisation of incorporated BrdU and KIN17 protein in adult testis. (A,D) BrdU staining appears green. (B,E) KIN17 staining appears red. (C,F) merged images where the colocalisation of KIN17 protein and BrdU appears yellow. (F) shows a magnification of the spermatogonia in merged images of the indicated area in D and E. Stages of the seminiferous epithelium are indicated with Roman numerals. Preleptotene spermatocytes: S, spermatogonia: Sg. Bars: A, B, C, D and E, 32 µm; F, 13 µm.

 


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  		Fig. 4. Detection of KIN17 protein by immunofluorescence on testicular cell spreads. (A,B) 2-day-old (C) 8-day-old and (D-F) adult mice cells. In neonatal testes, germ cell interphase nuclei are much larger (arrow in A) than somatic nuclei (B). Both germ and somatic interphase nuclei are stained positive (red) for KIN17 all over the nucleoplasm with stronger staining of large intranuclear structures (* in A and B). Germ and somatic metaphase chromosomes also exhibit different shapes and, in the same preparation, germ cell metaphases are well spread (B) compared to Sertoli cell metaphases (A). In both cell types staining is not detected at pericentromeric heterochromatic regions as shown after counterstaining with DAPI (blue, arrows in B and C). (C) In 8-day-old mice spermatogonia metaphase chromosomes (with premature centromeric region separation as previously described) (Bernardino-Sgherri et al., 2002Go) exhibit the same staining pattern. At different stages of adult germ cell, chromatin of pachytene (D), metaphase I (E) and metaphase II (F) spermatocytes is stained by anti-KIN17 antibodies, whereas reduced staining is observed at pericentromeric regions (arrows in E and F). Bars, 10 µm.

 


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  		Fig. 6. Mouse KIN17 protein associates with DNA and RNA in testes. (A) In vivo UVC cross-linking of DNA to KIN17 in testicular cells. Suspensions of freshly isolated testicular cells were irradiated with UVC. Cells were then lysed and treated with DNase I. KIN17 protein was immunoprecipitated with rabbit anti-KIN17 antibody (lanes 1 and 3) or pre-immune serum (PIS) (lane 2, control) (lower panel). The immune complex was end-labelled with [{gamma}-32P]ATP and T4 polynucleotide kinase and resolved on a 10% SDS-PAGE gel. The autoradiogram is shown in the upper panel. (B) The labelled material migrating at the position of KIN17 protein was excised from the gel, digested with proteinase K, subsequently re-digested with DNaseI and resolved by 10% PAGE in 8 M urea; the autoradiogram of this gel is shown. A labelled smear is observed only in lysates from UVC cross-linked cells and mainly in the lane containing the immunoprecipitation with anti-KIN17 polyclonal antibody. The deproteinated labelled polynucleotide that had been cross-linked to KIN17 protein is sensitive to degradation by DNase 1. (C) In vivo UVC cross-linking of RNA species to KIN17 in testicular cells. The cells were treated as above excepting that RNase A was used instead of DNase 1. (D) Note that the deproteinated labelled polynucleotide that had been cross-linked to KIN17 is sensitive to degradation by RNaseA.

 


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  		Fig. 7. KIN17 protein co-purifies with poly(A+) RNA. Extracts from mouse testicular cells were subjected to oligo (dT) chromatography under native conditions. Aliquots from total cell extract (lane 1), flow-through (lane 2), high-salt (lane 3) and low-salt (lane 4) washes and eluate (lane 5) fractions were subjected to SDS-PAGE and analysed by western blot. Previous RNase treatment of cell extracts abolishes KIN17, HUR and DAX-1 fractionation in the eluate (lanes 5 and 6).

 


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  		Fig. 8. KIN17 protein binds to RNA. (A) RNA homopolymer binding assay. Binding to agarose beads coupled to poly(A), poly(C), poly(G) and poly(U) is shown for wild-type mouse KIN17 protein, modified proteins KIN17335{Delta}KOW and KIN17160 translated in a rabbit reticulocyte lysate. The binding of human KIN17 protein expressed in insect cells to RNA homopolymers and detected by western blot is also shown. A 1:3 ratio of the input protein is shown in each case. The binding of protein to homopolymers was evaluated by radioactivity detection using STORM phosphoimager and immunodetection with the exception of KIN17160 protein that was not recognized by the mAb. Salt sensitivity of the binding was tested in buffers containing 0.1, 0.2, 0.5 and 1 M NaCl. (B) Northwestern binding assay using a [32P]-labelled poly(G) or (C) [32P]-labelled RNA probed against the proteins blotted on a nitrocellulose membrane. PSMW, prestained molecular weight markers, 1 µg of each protein was loaded.

 


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  		Fig. 9. A fraction of KIN17 protein is associated with chromatin and nuclear matrix. Testicular nuclear matrix was prepared as described in the Materials and Methods. Proteins were sequentially extracted with 0.5% Triton X-100 (lane1), DNase I and 0.25 M (NH4)2 SO4 (chromatin, lane 2) and 2 M NaCl (lane 3); the remaining pellet was solubilised in 8 M urea buffer (nuclear matrix, lane 4). 50 µg of protein from each step of the extraction protocol were subjected to SDS-PAGE and immunoblotted with antibodies against KIN17 protein, PCNA or LAMIN B (Santa Cruz Biotechnology) or SF2/ASF (gift from A. Krainer).

 

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© The Company of Biologists Ltd 2004