X-linked microRNA genes that escape msci co-localize with RNA polymersase II foci in pachytene spermatocytes

Date
2015
Authors
Flores, Luis
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Abstract

RNA polymerase II (RNA pol II) is known to be responsible for transcription of most microRNA (miRNA) genes, a class of short non-coding RNA molecules which function in post-transcriptional (and possibly transcriptional) gene regulation. Studies have shown that when RNA pol II is inhibited there is a decrease in primary-miRNAs, which are the precursors of mature miRNAs. Normally, all X- and Y-linked genes are repressed in primary spermatocytes by the process of Meiotic Sex Chromosome Inactivation (MSCI). However, previous studies, based largely on measurements of steady state levels of miRNAs, have demonstrated that a number of X-linked miRNA genes appear to escape MSCI. If this is the case, RNA pol II should co-localize to the sites of de novo transcription of these X-linked miRNA transcripts as well as with the X-linked miRNA genes in primary spermatocytes. To test this hypothesis, we performed RNA fluorescence in situ hybridization (RNA-FISH) to detect specific transcripts and DNA fluorescence in situ hybridization (DNA-FISH) to detect specific gene loci in combination with immunofluorescence (IF) staining of RNA Pol II. Our results demonstrate co-localization of miRNA genes and nascent pri-miRNA transcripts with RNA pol II in cell spreads of spermatogenetic cells, particularly in pachytene spermatocytes where MSCI normally represses transcription of sex-linked genes. We also investigated the existence of discrete foci of transcription (aka "transcription factories") in primary spermatocytes. Our findings suggest that Type II and III X-linked miRNA gene loci and nascent transcripts do co-localize with RNA pol II and that transcription factories do exist in primary spermatocytes. These results provide direct visual confirmation of the report that Type II and III X-linked miRNA genes escape MSCI and co-localize with transcription factories in pachytene spermatocytes.

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Keywords
Meiotic Sex Chromosome Inactivation, microRNA, MSCI, pachytene spermatocyte, spermatogenesis
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Department
Integrative Biology