Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knockdown widely used HaloTag7 fusion proteins in a general fashion.

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

Imprinted genes play important roles in embryonic growth and development as well as in placental function. Many imprinted genes acquire their epigenetic marks during oocyte growth, and this period may be susceptible to epigenetic disruption following hormonal stimulation. Superovulation has been shown to affect growth and development of the embryo, but an effect on imprinted genes has not been shown in postimplantation embryos. In the present study, we examined the effect of superovulation/in vivo development or superovulation/3.5dpc (days post-coitum) embryo transfer on the allelic expression of Snrpn, Kcnq1ot1 and H19 in embryos and placentas at 9.5 days of gestation. Superovulation followed by in vivo development resulted in biallelic expression of Snrpn and H19 in 9.5dpc placentas while Kcnq1ot1 was not affected; in the embryos, there was normal monoallelic expression of the three imprinted genes. We did not observe significant DNA methylation perturbations in the differentially methylated regions of Snrpn or H19. Superovulation followed by embryo transfer at 3.5dpc resulted in biallelic expression of H19 in the placenta. The expression of an important growth factor closely linked to H19, Insulin-like growth factor-II, was increased in the placenta following superovulation with or without embryo transfer. These results show that both maternally and paternally methylated imprinted genes were affected, suggesting that superovulation compromises oocyte quality and interferes with the maintenance of imprinting during preimplantation development. Our findings contribute to the evidence that mechanisms for maintaining imprinting are less robust in trophectoderm-derived tissues, and have clinical implications for the screening of patients following assisted reproduction.

The Piwi protein subfamily is essential for Piwi-interacting RNA (piRNA) biogenesis, transposon silencing, and germ-line development, all of which have been proposed to require Piwi endonuclease activity, as validated for two cytoplasmic Piwi proteins in mice. However, recent evidence has led to questioning of the generality of this mechanism for the Piwi members that reside in the nucleus. Drosophila offers a distinct opportunity to study the function of nuclear Piwi proteins because, among three Drosophila Piwi proteins-called Piwi, Aubergine, and Argonaute 3-Piwi is the only member of this subfamily that is localized in the nucleus and expressed in both germ-line and somatic cells in the gonad, where it is responsible for piRNA biogenesis and regulatory functions essential for fertility. In this study, we demonstrate beyond doubt that the slicer activity of Piwi is not required for any known functions in vivo. We show that, in transgenic flies with the DDX catalytic triad of PIWI mutated, neither primary nor secondary piRNA biogenesis is detectably affected, transposons remain repressed, and fertility is normal. Our observations demonstrate that the mechanism of Piwi is independent of its in vitro endonuclease activity. Instead, it is consistent with the alternative mode of Piwi function as a molecule involved in the piRNA-directed guidance of epigenetic factors to chromatin.small RNA | RNase H fold | transposable element | sterility