Identifying mRNAs Bound by Human RBMY Protein in the Testis

Abstract

Abstract. Rbmy gene encodes a germ-cell specific nuclear RNA-binding protein and is involved in spermatogenesis. To further investigate the specific events of spermatogenesis in which Rbmy plays a role, the target mRNAs of human RBMY protein were isolated and identified. Through the isolating specific nucleic acids associated with proteins (SNAAP) technique, we isolated twenty potential target genes of human RBMY protein from the human testis in the present study. Some of these target genes play important roles during spermatogenesis and have alternative transcripts in the testis. In this study, we focused on the human-related (never in mitosis gene a) kinase 10 (Nek10) gene, which belongs to the Nek protein kinase subfamily. Nek10 has two transcripts, and the results of RT-PCR and Electrophoretic Mobility Shift Assays (EMSA) show that hRBMY protein can only bind to transcript variant 2 of Nek10 and that hRbmy may take part in alternative splicing of Nek10. Isolation and identification of target genes of hRBMY will be helpful to further investigate the biological function of RBMY in spermatogenesis. Key words: Nek10, Rbmy gene, RNA-binding protein, SNAAP technique, Spermatogenesis (J. Reprod. Dev. 57: [107][108][109][110][111][112] 2011) permatogenesis is a complex cell differentiation process that requires highly regulated expression of lots of genes [1][2][3]. Many genes are mainly or exclusively expressed in the testis, and in most situations, further research has shown that they are only expressed in the germline cells [4]. Microdeletions at a specific region called the AZF in the long arm of Y chromosome have been described in significant proportions of oligo and azoospermic patients, suggesting that some genes located at these regions play an essential role during spermatogenesis [5]. Of all the genes located at the AZF region, Rbmy was the first of these candidate genes to be identified [6].There are many Rbm genes distributed throughout the Y chromosome, but only the genes in deletion interval AZFb give rise to detectable levels of protein [7]. According to the complete sequence of the human Y chromosome, it is now known that there are six Rbmy copies mapped in the AZFb region, but only one copy is actively transcribed [8]. Human and mouse Rbmy genes have a homologue on the human X chromosome (Rbmx) that maintains a widespread function, while Rbmy evolves a male-specific function in spermatogenesis [9]. There is another retrogene called hnRNPG-T that belongs to the same family as Rbmy and Rbmx, which is also specifically expressed in the testis. Previous study has shown that haploinsufficiency of hnRNPG-T results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of hnRNPG-T may be a cause of male infertility in humans [10]. Although Rbmy is evolutionarily conserved in the human, mouse and marsupial [11][12][13], their expression patterns are not completely identical. The expression of mouse Rbmy is limited to spermatogonia and early spermatocytes, while human Rbmy ...

Loss of maternal Trim28 causes male-predominant early embryonic lethality

“…S9). Targeting its own mRNA in testis for alternative splicing (Zeng et al 2008(Zeng et al , 2011, these severe defects may be attributed to expression of RBMY1A1 in the unnatural embryonic environment. Indeed, analysis of RNA sequencing (RNA-seq) reads revealed a number of gene products with exon-skipping events, which was validated for three genes (Scamp4, Mettl6, and Mybl2) by RT-PCR in individual embryos of both sexes and genotypes (Supplemental Table 4; Supplemental Fig.…”

Potential dual functional roles of the Y‐linked RBMY in hepatocarcinogenesis

“…RBMY protein harbors an RNA recognition motif (RRM) and functions as a regulator of germ cell‐specific splicing events 24‐27 . Although it shares a conserved RRM domain with its X homolog RBMX, they diverged at their flanking sequences.…”

“…RBMY is predominantly expressed in the male germ cells under normal conditions 15,23 . The encoded protein harbors an RNA‐binding motif and could participate in RNA splicing events in germ cells of the testis 24‐27 . Deletions in the RBMY genes cause failure in male meiosis, resulting in the absence of mature sperms in the testes of infertile patients 23 .…”

GDNF Signaling Regulates YBX1/mRNA Interactions in Mouse Spermatogonia.

“…This well conserved protein is also linked to male infertility, and it specifically associates with the mRNA of proteins necessary for spermatogenesis further establishing the role of RMBY1A1 and male germline specific splicing as critical to spermatogenic function [139][140][141]. Identifying other mRNA regulatory proteins, and their target mRNAs, will extend our basic biological understanding of SSCs and spermatogenesis beyond the transcriptome.…”

“…It further is an attractive candidate for clinical study of male infertility, because mutations in its genomic locus are associated with oliogospermgia and sperm abnormalities [137, 139]. Known for its role in mRNA splicing, studies in both mouse and human have identified RBMY1A1 target transcripts, many of which are alternatively spliced and some of which are testis specific, such as Tex14 [140,141]. These data imply a splicing role for RBMY1A1 in spermatogonia, and again, validate the study design.…”