論文

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.

Primary piRNAs in Drosophila ovarian somatic cells arise from piRNA cluster transcripts and the 3' UTRs of a subset of mRNAs, including Traffic jam (Tj) mRNA. However, it is unclear how these RNAs are determined as primary piRNA sources. Here, we identify a cis-acting 100-nt fragment in the Tj 3' UTR that is sufficient for producing artificial piRNAs from unintegrated DNA. These artificial piRNAs were effective in endogenous gene transcriptional silencing. Yb, a core component of primary piRNA biogenesis center Yb bodies, directly bound the Tj-cis element. Disruption of this interaction markedly reduced piRNA production. Thus, Yb is the trans-acting partner of the Tj-cis element. Yb-CLIP revealed that Yb binding correlated with somatic piRNA production but Tj-cis element downstream sequences produced few artificial piRNAs. We thus propose that Yb determines primary piRNA sources through two modes of action: primary binding to cis elements to specify substrates and secondary binding to downstream regions to increase diversity in piRNA populations.

MicroRNAs (miRNAs) are members of the small non-coding RNAs, which are principally known for their functions as post-transcriptional regulators of target genes. Regulation by miRNAs is triggered by the translational repression or degradation of their complementary target messenger RNAs (mRNAs). The growing number of reported miRNAs and the estimate that hundreds or thousands of genes are regulated by them suggest a magnificent gene regulatory network in which these molecules are embedded. Indeed, recent reports have suggested critical roles for miRNAs in various biological functions, such as cell differentiation, development, oncogenesis, and the immune responses, which are mediated by systems-wide changes in gene expression profiles. Therefore, it is essential to analyze this complex regulatory network at the transcriptome and proteome levels, which should be possible with approaches that include both high-throughput experiments and computational methodologies. Here, we introduce several systems-level approaches that have been applied to miRNA research, and discuss their potential to reveal miRNA-guided gene regulatory systems and their impacts on biological functions.

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

PIWI-interacting RNAs (piRNAs), a subset of small non-coding RNAs enriched in animal gonads, repress transposons by assembling with PIWI proteins to form potent gene-silencing RNP complexes, piRISCs. Accumulating evidence suggests that piRNAs are produced through three interdependent pathways; the de novo primary pathway, the ping-pong pathway, and the phased primary pathway. The de novo primary pathway in Drosophila ovaries produces primary piRNAs for two PIWI members, Piwi and Aub. Aub then initiates the ping-pong pathway to produce secondary piRNAs for AGO3. AGO3-slicer dependent cleavage subsequently produces secondary piRNAs for Aub. Trailer products of AGO3-slicer activity are consumed by the phased primary pathway to increase the Piwi-bound piRNA population. All these pathways are regulated by a number of piRNA factors in a highly coordinated fashion. Recent studies show that two Tudor-domain containing piRNA factors, Krimper (Krimp) and Qin/Kumo, play crucial roles in making Aub-AGO3 heterotypic ping-pong robust. This maintains the levels of piRNAs loaded onto Piwi and Aub to efficiently repress transposons at transcriptional and post-transcriptional levels, respectively.

PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. We suggest that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells.