Cloning and characterization of microRNAs from rainbow trout (Oncorhynchus mykiss): their expression during early embryonic development.
BACKGROUND
Current literature and our previous 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). MicroRNAs (miRNAs) are small, non-coding regulatory RNAs (19-23 nucleotides) that regulate gene expression by guiding target mRNA cleavage or translational inhibition. These regulatory RNAs are potentially involved in the degradation of maternally inherited mRNAs during early embryogenesis.


RESULTS
To identify miRNAs that might be important for early embryogenesis in rainbow trout, we constructed a miRNA library from a pool of unfertilized eggs and early stage embryos. Sequence analysis of random clones from the library identified 14 miRNAs, 4 of which are novel to rainbow trout. Real-time PCR was used to measure the expression of all cloned miRNAs during embryonic development. Four distinct expression patterns were observed and some miRNAs showed up-regulated expression during EGA. Analysis of tissue distribution of these miRNAs showed that some are present ubiquitously, while others are differentially expressed among different tissues. We also analyzed the expression patterns of Dicer, the enzyme required for the processing of miRNAs and Stat3, a transcription factor involved in activating the transcription of miR-21. Dicer is abundantly expressed during EGA and Stat3 is up-regulated before the onset of EGA.


CONCLUSIONS
This study led to the discovery of 14 rainbow trout miRNAs. Our data support the notion that Dicer processes miRNAs and Stat3 induces expression of miR-21 and possibly other miRNAs during EGA. These miRNAs in turn guide maternal mRNAs for degradation, which is required for normal embryonic development.
Ramachandra
Raghuveer K
RK
Laboratory of Animal Biotechnology and Genomics, Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV 26506, USA. raghuveerkr@gmail.com
Salem
Mohamed
M
Gahr
Scott
S
Rexroad
Caird E
CE
Yao
Jianbo
J
D000704:Analysis of Variance; D000818:Animals; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000196:embryology; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D035683:MicroRNAs / Q000235:genetics*; D017686:Oncorhynchus mykiss / Q000196:embryology / Q000235:genetics*; D012313:RNA; D012333:RNA, Messenger / Q000235:genetics; D017423:Sequence Analysis, RNA; D014018:Tissue Distribution
D005819:Genetic Markers; D035683:MicroRNAs; C034477:RNA primers; D012333:RNA, Messenger; D012313:RNA