Spermatid perinuclear ribonucleic acid-binding protein binds microtubules in vitro and associates with abnormal manchettes in vivo in mice.

Abstract

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The RNA target of SPNR in vivo is unknown, although we have previously suggested the possibility that SPNR is involved in the translational activation of the protamine 1 mRNA in elongated spermatids. To increase our understanding of SPNR's association with the manchette, we sought to determine SPNR's subcellular localization in several mouse mutants that show reduced fertility or sterility and that have structurally abnormal manchettes. We show here that despite the highly abnormal manchettes and microtubule aggregates formed in azh, hop-sterile, tw2, and tw8 mutants, SPNR remains associated with the manchettes. Localization of SPNR to the abnormal manchettes suggests that SPNR is tightly bound to the manchette. SPNR could bind manchette microtubules directly, or it could bind indirectly via an interaction with a microtubule-associated protein (MAP). We sought to determine whether SPNR binds microtubules in vitro, and if so, whether it requires a MAP. We show by Western analysis that the endogenous SPNR protein can be pelleted with murine testis microtubules in a taxol-dependent manner in vitro. A recombinant version of SPNR produced in bacteria can also be pelleted with testis microtubules, as well as microtubules polymerized from purified bovine brain tubulin, an association that is salt-sensitive. These results suggest that SPNR, in addition to its function as an RNA-binding protein, is also a bona fide MAP.