Cutoff Suppresses RNA Polymerase II Termination to Ensure Expression of piRNA Precursors.

Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

Small but sturdy: small RNAs in cellular memory and epigenetics.

Cells in multicellular organisms have distinct identities characterized by their profiles of expressed genes. Cell identities can be stable over a long time and through multiple cellular divisions but are also responsive to extracellular signals. Since the DNA sequence is identical in all cells, a "cellular memory" of expression profiles is achieved by what are defined as epigenetic mechanisms. Two major molecular principles--networks of transcription factors and maintenance of cis-chromatin modifications--have been implicated in maintaining cellular memory. Here we describe recent studies demonstrating that short noncoding RNAs can also provide molecular signals that define epigenetic states of cells. Small RNAs can act independently or cooperate with chromatin modifications to achieve long-lasting effects necessary for cellular memory and transgenerational inheritance.

Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing.

Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors.

The piRNA Pathway Guards the Germline Genome Against Transposable Elements.

Transposable elements (TEs) have the capacity to replicate and insert into new genomic locations. This contributs significantly to evolution of genomes, but can also result in DNA breaks and illegitimate recombination, and therefore poses a significant threat to genomic integrity. Excess damage to the germ cell genome results in sterility. A specific RNA silencing pathway, termed the piRNA pathway operates in germ cells of animals to control TE activity. At the core of the piRNA pathway is a ribonucleoprotein complex consisting of a small RNA, called piRNA, and a protein from the PIWI subfamily of Argonaute nucleases. The piRNA pathway relies on the specificity provided by the piRNA sequence to recognize complementary TE targets, while effector functions are provided by the PIWI protein. PIWI-piRNA complexes silence TEs both at the transcriptional level - by attracting repressive chromatin modifications to genomic targets - and at the posttranscriptional level - by cleaving TE transcripts in the cytoplasm. Impairment of the piRNA pathway leads to overexpression of TEs, significantly compromised genome structure and, invariably, germ cell death and sterility.The piRNA pathway is best understood in the fruit fly, Drosophila melanogaster, and in mouse. This Chapter gives an overview of current knowledge on piRNA biogenesis, and mechanistic details of both transcriptional and posttranscriptional TE silencing by the piRNA pathway. It further focuses on the importance of post-translational modifications and subcellular localization of the piRNA machinery. Finally, it provides a brief description of analogous pathways in other systems.

Structural and functional analysis of the C-terminal domain of Nup358/RanBP2.

The nuclear pore complex is the sole mediator of bidirectional transport between the nucleus and cytoplasm. Nup358 is a metazoan-specific nucleoporin that localizes to the cytoplasmic filaments and provides several binding sites for the mobile nucleocytoplasmic transport machinery. Here we present the crystal structure of the C-terminal domain (CTD) of Nup358 at 1.75Å resolution. The structure reveals that the CTD adopts a cyclophilin-like fold with a non-canonical active-site configuration. We determined biochemically that the CTD possesses weak peptidyl-prolyl isomerase activity and show that the active-site cavity mediates a weak association with the human immunodeficiency virus-1 capsid protein, supporting its role in viral infection. Overall, the surface is evolutionarily conserved, suggesting that the CTD serves as a protein-protein interaction platform. However, we demonstrate that the CTD is dispensable for nuclear envelope localization of Nup358, suggesting that the CTD does not interact with other nucleoporins.