In this project a block in processing of primary miRNA transcripts (pri-miRNAs) in human embryonic stem cells was demonstrated. Pri-miRNAs for 15 out of 24 tested intergenic miRNAs were detected by RT-PCR in the H1 cell line despite a lack of detection of their mature form in deep sequencing data. By contrast, only 2 out of 16 pri-miRNAs for which expression of the mature form was not reported were successfully amplified by RT-PCR in HEK293T cells. A reduction in the pri-miRNA levels was observed upon blocking transcription in H1 cells and the vast majority of the same pri-miRNAs were detected by RT-PCR using nascent RNA, indicative of their active transcription and degradation.
To test whether hESCs cells vigorously transcribe yet degrade more mRNA transcripts in general than differentiated cells we isolated nascent and total RNA samples from H1 and HEK293T cells and performed a microarray analysis using the Illumina HT-12 v4 platform. There were no significant differences in the number of genes transcribed in the two cell lines and across both types of RNA samples. These results suggested that a similar proportion of the coding part of the genome is actively or vigorously transcribed in both hESCs and differentiated cell types.
It is generally thought that ESCs have a higher level of background transcription than normal, due to the more open chromatin in this cell type. We therefore suggest that background transcription might be responsible for our detection of immature miRNA transcripts in hESCs instead of vigorous transcription and that enhanced background transcription has a measurable impact perhaps because pri-miRNAs are normally only transcribed at relatively low rates compared to other genes. We still need to explain why some pri-miRNA transcripts are degraded in ESCs. One possibility is that such miRNAs express both sense and antisense transcripts.