MIWI-independent small RNAs (MSY-RNAs) bind to the RNA-binding protein, MSY2, in male germ cells

Abstract

The germ cell-specific DNA/RNA-binding protein MSY2 binds small RNAs (MSY-RNAs) that are Ϸ25-31 nt in length, often initiate with a 5 adenine, and are expressed in both germ cells and somatic cells. MSY-RNA levels do not decrease in Miwi or Msy2 null mice. Most MSY-RNAs map within annotated genes, but some are PIWIinteracting RNA (piRNA)-like and map to piRNA clusters. MSY-RNAs are in both nuclei and cytoplasm. In nuclei, MSY-RNAs are enriched in chromatin, and in the cytoplasm they are detected in both ribonucleoproteins and polysomes.noncoding RNA ͉ RNA-protein interactions ͉ Y box proteins ͉ spermatogenesis N oncoding RNAs play important roles in regulating gene expression and development (1). Among the many classes of small noncoding RNAs are micro RNAs (miRNAs), siRNAs, endo-siRNAs, and PIWI-interacting RNAs (piRNAs) (reviewed in refs. 2-4). piRNAs are an extremely abundant group of germ cell RNAs of Ϸ30 nt that often initiate with a 5Ј uracil and require members of the PIWI family of proteins for their biogenesis (4, 5). Based on sequence and their time of expression, piRNAs in mice have been classified as MILI (PIWIL2)-associated, MIWI2 (PIWIL4)-associated, or MIWI (PIWIL1)-associated piRNAs (4, 6). They are believed to be transcribed from long precursor RNAs and processed by a Dicerindependent mechanism (4, 6). Although some mammalian piRNAs share features with the Drosophila repeat-sequence repeat associated small interfering RNA (rasiRNAs) that are proposed to control transposable elements (6-8), the large number, variable sequences, and nuclear localization of mammalian piRNAs suggest additional cellular functions (9, 10).The germ cell-specific DNA/RNA-binding protein MSY2 is a member of a Y box family of cold shock domain proteins (11). It is solely expressed in male and female germ cells and necessary for fertility (12). In the male, the absence of MSY2 leads to precocious destabilization of germ cell mRNAs (12). MSY2 serves as a coactivator of transcription, binding to the DNA sequence CTGATTGGC/TC/TAA in the promoters of genes transcribed in germ cells, thereby linking transcription with mRNA storage/stabilization (11). Suppressive subtractive hybridization studies reveal that of the nearly 100 MSY2 bound/ nonbound mRNAs analyzed, many mRNAs encoding male gamete-specific and translationally delayed proteins are bound by MSY2 (11). In addition, MSY2 also binds in vitro to the piRNA, gsRNA10 (13).Here, we describe a previously uncharacterized class of small RNAs (MSY-RNAs) from the mouse testis that are bound by MSY2. MSY-RNAs are Ϸ25-31 nt, often initiate with a 5Ј adenine, and are expressed throughout germ cell differentiation and in somatic cells. The majority of the MSY-RNAs are derived from annotated genes, and some MSY-RNAs are piRNAs or piRNA-like. MSY-RNA levels are not altered in MIWI or MSY2 null mice. Results and Discussion MSY2 Selectively Binds a Previously Uncharacterized Population ofϷ30-Nucleotide RNAs. Because in vitro RNA gel shift assays revealed that MSY2 binds to the piRNA, gsRN...

Widespread expression of piRNA-like molecules in somatic tissues

“…Rabbit anti-Miwi antibody raised against the peptide VIRVPAPCQYAHKLAFLVGQ (amino acids 826–845) and mouse anti-actin antibody (Sigma) were used for immunoblotting as described in (42). …”

HITS‐CLIP: panoramic views of protein–RNA regulation in living cells

“…Although the properties of MSY2 as an mRNA binding protein have previously been studied, recent work suggested that it might also bind small RNAs. Therefore a "directed-CLIP" experiment was done in which small RNA-protein complexes were isolated on the basis of elecrophoretic mobility 91 . 231 CLIP tags were sequenced and found to be small RNAs expressed in testes ranging in length from 18 to 36 nt.…”

RNA–protein interactions in human health and disease

“…MSY2, a germ-cell-specific member of the Y-box family of DNA-/RNA-binding proteins, have recently been shown to associate with a novel class of small RNAs. MSY2 binds small RNAs (MSY-RNAs) that range in size from 18–36 nt with the majority being 25–31 nt in length, and are expressed in both germ cells and somatic cells [54]. Despite the abundance of piRNAs in the testes which are also similar in size to the immunoprecipitated RNAs with MSY; the authors found only 7% of these MSY-RNA map to known piRNA genes.…”