Biology subjects

Mowla, S. J.

Publications and source records attributed to Mowla, S. J..

3 recordsLinked to original sources

bioRxiv2018

Expression of the miR-302/367 microRNA cluster is regulated by a conserved long non-coding host gene

MicroRNAs (miRNAs) are important regulators of cellular functions. MiR-302/367 is a polycistronic miRNA cluster including miR-302b/c/a/d (collectively termed miR-302s) and miR-367. The cluster is located in the intron of a non-coding host gene. MiR-302s have been shown to repress mRNAs required for differentiation and to induce pluripotency in somatic cells. The stem cell specific transcription factors OCT4, SOX2 and Nanog drive miR-302s expression, however, the reported expression in human and mice indicates a more complex transcriptional regulation. Here we investigate the transcriptional control and the processing of the miR-302 host gene. The murine miR-302 host gene is alternatively spliced, polyadenylated and exported from the nucleus. The regulatory sequences extend at least 2 kb upstream of the transcription start side and contain several conserved binding sites for both transcriptional activators and repressors. Reporter constructs with different upstream regions revealed a significant influence of the more distant regulatory sequences in pluripotent stem cells. The gene structure and regulatory elements like binding sites for activating and repressing transcriptional regulators, splice, and polyadenylation signals are highly conserved between mouse and human. So far, no miR-302 independent function has been annotated for the miR-302 host gene and we hypothesize that the complex and differential regulation of the miRNA transcription and processing might the reason for its conservation. Thus, regulation or micro-RNA expression might be a so far less recognized function of non-coding RNA genes.\n\nAuthor SummaryNon-coding RNAs constitute a large part of the mammalian genome. Interestingly, some long non-coding RNAs (lncRNA) are transcribed and processed in the same way as mRNAs but lack an open reading frame. Here we give evidence that a so far less recognized function of such lncRNAs could be to supply microRNAs with the complex transcriptional control and processing required for their intricate expression. As an example, we analyzed the regulatory sequences of the miR-302/367 host gene. MiR-302/367 is a microRNA cluster involved in the regulation of stem cells and cellular differentiation. We show here that the regulatory region is much more complex than anticipated, a complexity that can not be conferred alone by any of the stem cell specific transcription factors which were so far associated with the expression of miR-302/367.

genetics↗

bioRxiv2018

Transgenic Mice and Pluripotent Stem Cells Express EGFP under the Control of miR-302 Promoter

MicroRNAs are a group of short non-coding RNAs that undertake various roles in different cell signaling pathways and developmental stages. They regulate gene expression levels at the post-transcriptional stage, which results in cleavage of mRNAs or repression of their translation. Some miRNAs, including the miR-302 cluster, are critical regulators for the stemness state of embryonic stem cells and cell fate patterning. The miR-302 cluster is located in the intron of a non-coding gene that has no other reported function, other than hosting miR-302, and grant a complex expression regulation through upstream its regulatory sequences. To date, analysis of the miR-302 expression pattern in a transgenic mouse model has not been reported. In this study, we generated transgenic mice that expressed EGFP driven by miR-302 upstream regulatory sequences that harbored the core promoter of its host gene. We examined the activity of the miR-302 promotor in somatic tissues of transgenic mice, transgenic blastocysts, and embryonic stem cells derived from transgenic blastocysts. Our results showed that miR-302 highly expressed in both blastocysts and the first passages of transgenic embryonic stem cells, and has low expression in the somatic tissues of transgenic mice. It could be concluded that different temporal and spatial gene expression patterns occur during the embryonic and adult stages in mice.

developmental biology↗

bioRxiv2018

Isolation of cancer stem cells by selection for miR-302 expressing cells

Cancer stem cells receive increasing interest because they are believed to be a major reason for long-term therapy failure. The reason for the therapy resistance of cancer stem cells lies partially in their multi-drug resistance and partially in the ability to rest mitotically inactive in the hypoxic center of tumors. Due to their variable number and their often low proliferation rate, cancer stem cells are difficult to purify in decent quantities and to grow in cell culture systems, where they are easily outcompeted by faster growing more differentiated, i.e. less stem cell-like tumor cells. Here we present a proof of principle study based on the idea to select cancer stem cells by means of the expression of a stem cell-specific gene. We inserted a selectable egfp-neo coding sequence in the last exon of the non-coding murine miR-302 host gene. As a stem cell specific regulatory element, we used 2.1 kb of the genomic region immediately upstream of the miR-302 host gene transcription start. Stable transgenic CJ7 embryonic stem cells were used to induce teratomas. After three weeks, tumors were removed for analysis and primary cultures were established. Stem-like cells were selected from these culture based on G418 selection. When the selection was removed, stem cell morphology and miR-302 expression were rapidly lost, indicating that it were not the original ES cells that have been isolated. In conclusion, we show the possibility to use drug resistance expressed from a regulatory sequence of a stem cell-specific marker, to isolate and propagate cancer stem cells that otherwise might be hidden in the majority of tumor cells.

cancer biology↗