KIN17 encodes an RNA-binding protein and is expressed during mouse spermatogenesis

Abstract

Genotoxic agents deform DNA structure thus eliciting a complex genetic response allowing recovery and cell survival. The Kin17 gene is up-regulated during this response. This gene encodes a conserved nuclear protein that shares a DNA-binding domain with the bacterial RecA protein. The KIN17 protein binds DNA and displays enhanced expression levels in proliferating cultured cells, suggesting a role in nuclear metabolism. We investigated this by studying the expression profile of KIN17 protein during mouse spermatogenesis. As expected, the expression level of Kin17 is higher in proliferating than in differentiated cells. KIN17 is selectively extracted from this tissue by detergents and a fraction was tightly associated with the nuclear matrix. Germinal cells ubiquitously express Kin17 and the protein is located mainly in the nucleus except in elongated spermatids where cytoplasmic staining is also observed. Sertoli and germ cells that are no longer mitotically active express KIN17, suggesting a general role in all testicular cell types. In adult testis a significant proportion of KIN17 co-purifies with polyadenylated RNA. KIN17 directly binds RNA, preferentially poly(G) and poly(U) homopolymers. These results together with the identification of KIN17 as a component of the human spliceosome indicate that this protein may participate in RNA processing.

A Tandem of SH3-like Domains Participates in RNA Binding in KIN17, a Human Protein Activated in Response to Genotoxics

“…Thus, the C-terminal domain of hKIN17 has structural similarity with various SH3-like domains, even if it lacks similarities in either primary sequence or charge distribution. The C-terminal domain of hKIN17 binds RNA in vitro Since hKIN17 protein is able to recognize RNA homopolymers and can bind RNA in vivo, 16 we investigated which region(s) of the protein interact(s) with RNA. Three constructs containing either the Nterminal globular region 1-160 (shown to be folded by NMR analysis; data not shown), the same region without the zinc finger 68-160 (shown to be folded by NMR analysis), 24 or the C-terminal domain 268-393 described here were tested for their ability to bind RNA using the same 32 P-labelled RNA probe as that used to characterize the RNA interaction properties of the full-length hKIN17.…”

“…Three constructs containing either the Nterminal globular region 1-160 (shown to be folded by NMR analysis; data not shown), the same region without the zinc finger 68-160 (shown to be folded by NMR analysis), 24 or the C-terminal domain 268-393 described here were tested for their ability to bind RNA using the same 32 P-labelled RNA probe as that used to characterize the RNA interaction properties of the full-length hKIN17. 16 The Northwestern analysis showed that the N-terminal domain comprising the zinc finger interacts efficiently with RNA in vitro, and that the deletion of the zinc finger from this domain drastically reduces RNA binding ( Figure 3). The C-terminal domain showed an intermediate RNA-binding activity.…”

“…16 To allow renaturation of the recombinant proteins after they were separated by SDS-PAGE and transferred onto nitrocellulose membranes, the protein blots were treated for 1-2 h at room temperature in a hybridization buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1X Denhardt's solution, 1 mM EDTA) in the presence of 1 μg/ml of E. coli tRNA (Sigma). The blots were then probed at room temperature for 1 h with radiolabelled RNA probe (100,000 cpm for a 2 ml reaction volume) in a hybridization buffer containing 2 μg/ml of E. coli tRNA.…”

“…Binding of the purified recombinant protein to RNA homopolymers was performed as described. 16 A portion (20 μl) of each RNA homopolymer bead was incubated with an equal amount of purified protein (3 μg), for 30 min at 4°C with gentle agitation in a total volume of 500 μl of binding buffer (20 mM Tris-HCl (pH 7.4), 2.5 mM MgCl 2 , 0.5% (v/v) Triton X-100 and 100 mM, 250 mM, 500 mM or 1 M NaCl. After incubation, the beads were washed five times with 500 μl of binding buffer.…”

“…13 Moreover, proteomic analysis detected protein KIN17 in several complexes of the human spliceosome, a large RNAprotein assembly consisting of small ribonucleoprotein particles (snRNP) and associated non-small ribo-nucleoproteins. 14,15 Interaction between KIN17 and RNA was further characterized by Pinon-Lataillade et al, 16 showing that the mouse KIN17 protein interacts with RNA in vivo and that human and mouse KIN17 proteins are able to directly bind different types of RNA homopolymers in vitro.…”