Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): their expression during early embryonic development

Authors: RAMACHANDRA, RAGHUVEER - WEST VIRGINIA UNIVERSITY; SALEM, MOHAMED - WEST VIRGINIA UNIVERSTIY; Gahr, Scott; Rexroad, Caird; YAO, JIANBO - WEST VIRGINIA UNIVERSTIY

Submitted to: BMC Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2008
Publication Date: 4/15/2008
Citation: Ramachandra, R., Salem, M., Gahr, S.A., Rexroad III, C.E., Yao, J. 2008. Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): their expression during early embryonic development. Biomed Central (BMC) Genomics. 8:41.

Interpretive Summary: Understanding the processes driving embryonic development will help address issues related to reproduction and egg quality. After fertilization, embryos remain dependent on maternally stored mRNAs for their transcriptional needs until their own transcription machinery is functional. The timing of this transcriptional independence, called embryonic genome activation, varies greatly with species. In fishes this transition is characterized by asynchronous cell division, lengthening of cell cycles, cell motility and initiation of zygotic transcriptional machinery. After this activation, the embryo becomes increasingly dependent on its own transcription and exhausts the maternally stored mRNAs. It was recently shown that it is critical to degrade maternally inherited mRNAs to achieve normal development. Therefore there must be a tightly controlled regulatory mechanism in nature to degrade maternal mRNAs at the time of embryonic genome activation. Previously we showed that several transcription factors and oocyte specific genes are degraded during/around the time of embryonic genome activation in rainbow trout. Mechanisms by which this degradation is accomplished escaped scientific attention until recently. It was hypothesized by Schier that miRNAs are involved in these degradation processes. MicroRNAs are small, 19-23 bp non-coding RNAs that bind to their respective recognition sequences on mRNAs and identify them for degradation. Due to the lack of genomic information in rainbow trout, we designed this experiment to identify and characterize miRNAs potentially regulating maternal RNA degradation processes during embryonic development. Here we present data to show that expression of Dicer, the enzyme required for the processing of all miRNAs, and several miRNAs are synchronized at the time of maternal mRNA degradation. Signal transducer and activator of transcription3 (Stat3), which activates transcription of miRNAs, is also abundant in these stages indicating its role in inducing miRNA expression and thereby directing maternal mRNAs for degradation. This is the first report of microRNAs in rainbow trout and the first to examine its mechanisms of RNA degradation with respect to the embryonic genome activation.

Technical Abstract: Current literature and our results on expression patterns of oocyte specific genes and transcription factors suggest a global but highly regulated maternal mRNA degradation at the time of embryonic genome activation (EGA). We hypothesized that microRNAs (miRNAs), naturally occurring 19-21bp long post-transcriptional regulators, are involved in mRNA degradation processes during EGA. Results To identify candidate miRNAs involved in EGA, we constructed a miRNA library from a pool of oocytes and early embryonic stages (1-5 days post-fertilization, dpf). Sequencing analysis of clones identified 14 miRNAs expressed during these stages, 4 of which are novel to rainbow trout. Real-time PCR was used to measure their expression patterns during embryonic development and in adult tissues. Several miRNAs showed characteristic up-regulated expression patterns at the onset of maternal RNA degradation. We also analyzed the expression pattern of Dicer, an enzyme required for the processing of miRNAs and Stat3, a transcription factor which is involved in activating the transcription of miR-21. Dicer is abundantly expressed during of EGA and Stat3 was up-regulated before onset of the EGA. Conclusion Our data support the hypothesis that Dicer processes mature miRNAs during embryonic genome activation stages and these miRNAs in turn degrade maternal mRNAs. Stat3 induces expression of miR-21 and other miRNAs thereby guiding maternal mRNAs for degradation. Rainbow trout adult tissues exhibited characteristic miRNAs expression patterns, some of which are evolutionary conserved.