MicroRNAs (miRNAs) are small endogenous non-coding transcripts with diverse roles in development, differentiation and oncogenesis, which also play important roles in embryonic stem cells (ESCs). However, the miRNA expression profile in rhesus macaque ESCs is largely unknown.

We conducted microRNA profilings of three new rESC lines by Solexa sequencing. A total of 352 known miRNAs were identified with 304(92.4%) shared among the three rESC lines. We also identified 62 novel miRNA precursors (66 mature miRNAs) shared among the three lines. The miRNA profiles are highly conserved and consistent among the rESC lines, and their predicted target genes were significantly enriched in pathways involved in differentiation. Further analysis on miRNA-target network indicated that there was a negative correlation between evolutionary rate and degree of targets regulated by miRNAs in the rESCs. Moreover, the cross-species comparison of ESCs revealed an over-all conservation of miRNA expression patterns. However, three miRNA clusters (the miR-467cluster, the miRNA cluster in the imprinted Dlk1-Dio3 region and the C19MC cluster) showed sharp differences among mouse, rhesus macaque and human ESCs, suggesting that species and/or lineage specific miRNA regulatory changes also exit during evolution.

MiR-513 family is an X-linked miRNA cluster with mutiple copies and rapid evolution in primates, and is preferentially expressed in testis. Sequence comparisons revealed that the duplicated copies of miR-513 were derived from transposable elements (MER91C). Different copies of the miR-513 cluster (miR-513a, b, c) have diverse seed sequences, which may lead to functional divergence. Our results showed that miR-513b could inhibit the expression of its target gene, down-regulator of transcription 1 (DR1) at both mRNA and protein levels. Interestingly, a temporal coupling of expression levels was observed between miR-513b and DR1 in the developing testis of rhesus macaque. We propose that miR-513b would affect male sexual maturation by negatively regulating the development-stage-related functioning of DR1.