Minireview: The roles of small RNA pathways in reproductive medicine

Abstract

The discovery of small noncoding RNA, including P-element-induced wimpy testis-interacting RNA, small interfering RNA, and microRNA, has energized research in reproductive medicine. In the two decades since the identification of small RNA, first in Caenorhabditis elegans and then in other animals, scientists in many disciplines have made significant progress in elucidating their biology. A powerful battery of tools, including knockout mice and small RNA mimics and antagonists, has facilitated investigation into the functional roles and therapeutic potential of these small RNA pathways. Current data indicate that small RNA play significant roles in normal development and physiology and pathological conditions of the reproductive tracts of females and males. Biologically plausible mRNA targets for these microRNA are aggressively being discovered. The next phase of research will focus on elucidating the clinical utility of small RNA-selective agonists and antagonists.

Fig. 1.

piRNA biosynthesis. Two major pathways can generate piRNA. In primary piRNA biogenesis the PIWI protein, here MILI, cuts a precursor RNA into piRNA. In secondary piRNA biogenesis piRNAs in a sense orientation to active full-length copies of retrotransposon LINE1 (blue) bound to MILI target cleavage of transcripts containing fragments of LINE1 (3′-terminal fragment depicted as “NE1”) transcribed by antisense-oriented promotion. These new antisense piRNA can be incorporated in complex with MIWI2 to cleave the sense-oriented transcripts of LINE1 to generate more sense piPRNA to repeat the cycle.

Fig. 2.

Genetic model for nuage assembly and piRNA pathway genes in mice. Many of the proteins involved in piRNA production are conserved between Drosophila and the mouse (Table 2). Overall, two major types of piRNA granules are present: the MILI and MIWI granules are exclusively localized to the cytoplasm whereas the MIWI2 granules shuttle between the nucleus and cytoplasm. Whereas the cytoplasmic granule alone can produce primary piRNA (sense orientation), both granules are necessary for the production of secondary piRNA (antisense orientation) characteristic of the ping-pong amplification cycle. Three protein domains necessary for piRNA granule assembly that recur within the pathway components are the PIWI (green), TUDOR (red), and RNA helicase domains (blue). Mutual dependencies are represented by double-headed arrows. Components with insufficient evidence to assign a position within the pathway are listed at the bottom. See Table 2 for more detailed description of orthologous components.

Fig. 3.

A, A phylogenic comparison of the PIWI and AGO families of Argonaute RNA endonucleases in humans. Some mammalian clades (rodents) lack PIWIL3. B, Target overlap among multiple small RNA classes. DICER-dependent AGO-associated small RNA cause the degradation or translational repression of mRNA targets with endogenous siRNA regulating retrotransposons whereas miRNA control cellular mRNA. Small RNA associated with the PIWI subfamily of Argonautes have developed convergent roles to regulate retrotransposons (repeat-associated piRNA) and possibly cellular mRNA (non-repeat-associated piRNA). Genetic studies have delineated a sex-specific division of labor among the small RNA dedicated to retrotransposon surveillance in which endo-siRNA are the major player in the female germ cells whereas the male germ cell genome stability depends upon repeat-associated piRNA. nt, Nucleotides.

Fig. 4.

siRNA and miRNA biosynthesis. siRNA are initially transcribed from promoters within viral genomes, retrotransposons, adjacent genes with a tail-to-tail or overlapping head-to-head (data not shown) orientation of RNA promotion, or from pseudogene mRNA with antisense promotion paired to their functional gene paralogs. The resulting transcripts form a region of dsRNA. By contrast miRNA are transcribed from RNA pol II promoters intronic to other protein-encoding genes or under their own promoters and fold into a hairpin structure containing a region forming a dsRNA stem called a primary miRNA (pri-miRNA). The Microprocessor complex, composed of nuclear type III RNA endonuclease RNASEN/DROSHA and its cofactor DGCR8, excises the pre-miRNA by cleaving at the base of the pri-miRNA stem (orange triangles) and exported from the nucleus by exportin 5. Subsequently, the cytoplasmic type III RNA endonuclease DICER and cofactor TAR RNA binding protein 2 (TARBP2) cleaves the 5′-overhangs of the siRNA precursor or the loop of the pre-miRNA (red triangles). A mature siRNA or miRNA (single strand of the dsRNA) is loaded onto the Argonaute-containing effector complex, RNA-induced silencing complex. In Argonaute cocrystal structures the seed sequence is held rigidly between the PIWI and PAZ domains of the Argonaute allowing base pairing required for targeting. In mammals, AGO2 contains the conserved residues necessary for and demonstrated activity of endonucleolytic cleavage of target mRNA. After mRNA endonucleolytic cleavage, the target mRNA is decapped and exonuclease digestion occurs. The remaining Argonautes (AGO1, AGO3, and AGO4) lack the conserved endonuclease residues but may participate in the alternative outcome of miRNA action, translational repression.