Abstract
Distribution and localization of a RNA-binding protein, BRUNOL2 in rat spermatogenic cells were studied by dot blotting of cell fractions, immunofluorescence (IF), and immunoelectron microscopy (IEM). BRUNOL2 distributed in nuclear (23%), mitochondrial (19%), microsomal (15%), and cytosol fractions (43%). BRUNOL2 was detected in all spermatogenic cells. In the cytoplasm and nucleoplasm of the spermatogonia, spermatocytes and spermatids, both diffuse and granular staining patterns were observed. Many cytoplasmic granules were stained also for DDX4 and DDX25. Large granules in the cytoplasm of elongated spermatids were stained for BRUNOL2 but not for the nuage proteins. IEM showed that gold signals for BRUNOL2 were concentrated in nuage components including loose aggregates of small particles, chromatoid body (CB), intermitochondrial cement (IMC), and satellite body (SB). In addition, many non-nuage structures such as ER-attached small granules, less dense material surrounding connecting piece of flagellum, reticulated body, mitochondria-associated granules (MAG), granulated body, ribosome aggregate, and manchette, were stained for BRUNOL2 with different staining intensities. In the nucleus, gold signals were concentrated in heterochromatin area and nucleolus. The results suggest that BRUNOL2 is one of the nuage proteins and also associated with the other non-nuage structures, suggesting multiple functions of this protein.
Highlights
BRUNOL2 is a RNA-binding protein and belongs to CELF/BRUNO-like family
We study the co-localization of BRUNOL2 with known nuage proteins, DDX4 and DDX25, in rat spermatogenic cells by IF and immunoelectron microscopy (IEM) techniques
The 55 kDa band was observed in the cytosol fraction, which seemed to be one of BRUNOL2 isoforms, whereas 73 kDa band was detected in the mitochondrial fraction
Summary
BRUNOL2 is a RNA-binding protein and belongs to CELF/BRUNO-like family. Its major function in the nucleus is a regulatory role in the alternative splicing of target pre-mRNAs and in the control of translation and mRNA stability in the cytoplasm [1,2,3]. CELF/BRUNOlike protein, CUGBP1, and the related protein Etr-3 regulate the muscle-specific splicing of cardiac troponin T pre-mRNA [4,5]. The binding of these proteins to CUG repeats suggests that they play roles in myotonic dystrophy [4,6]. CELF1/BRUNO-like protein is localized both in the cytoplasm and the nucleus [6,7,8]. This dual localization suggests multiple functions of the protein. We study the co-localization of BRUNOL2 with known nuage proteins, DDX4 and DDX25, in rat spermatogenic cells by IF and IEM techniques
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