Visualization of de novo Gene Transcription of X-linked miRNAs that Escape Meiotic Sex Chromosome Inactivation

microRNAs (miRNAs) are a recently discovered class of non-coding RNAs that are known to regulate gene expression at the post-transcriptional level.  Studies in vivo using mice with gene knockouts of miRNAs have revealed that they are critically required for normal spermatogenesis and male fertility.  In a previous collaborative study, we found that ~20% of testicular miRNAs map to the X-chromosome and that ~40% of these X-linked miRNAs display testis-specific or testis preferential expression.  Surprisingly, real-time qPCR analysis further revealed an increase in the expression of the majority of these X-linked miRNA transcripts in spermatocytes, at a time when all 364 X-linked mRNA-encoding genes studied to date are seen to decline in expression due to the phenomenon of Meiotic Sex Chromosome Inactivation (MSCI).  We hypothesized that elevated levels of X-linked miRNA transcripts in primary spermatocytes are due to continued active transcription of these genes, indicating escape from the repressive effects of MSCI.  Using immunofluorescence staining and RNA-Fluorescence in-situ hybridization on spermatogenic cells from testes of adult mice, we have distinguished de novo transcription of a constitutively expressed autosomal gene (positive control) from that of an X-linked mRNA gene (negative control) subject to inactivation by MSCI.  Lastly, we have preliminary data indicating ongoing expression of an X-linked miRNA that escapes MSCI in spermatocytes.  These results provide the most direct data to date indicating that certain X-linked miRNA genes escape MSCI, and allow us to directly compare miRNA gene transcriptional activity with mRNA gene transcriptional inactivity due to MSCI.