Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

SUMMARYMaize anther ontogeny is complex, with the expression of more than 30 000 genes over 4 days of cell proliferation, cell fate acquisition and the start of meiosis. Although many male-sterile mutants disrupt these key steps, few have been investigated in detail. The terminal phenotypes of Zea mays (maize) male sterile 8 (ms8) are small anthers exhibiting meiotic failure. Here, we document much earlier defects: ms8 epidermal cells are normal in number but fail to elongate, and there are fewer, larger tapetal cells that retain, rather than secrete, their contents. ms8 meiocytes separate early, have extra space between them, occupied by excess callose, and the meiotic dyads abort. Thousands of transcriptome changes occur in ms8, including ectopic activation of genes not expressed in fertile siblings, failure to express some genes, differential expression compared with fertile siblings and about 40% of the differentially expressed transcripts appear precociously. There is a high correlation between mRNA accumulation assessed by microarray hybridization and quantitative real-time reverse transcriptase polymerase chain reaction. Sixty-three differentially expressed proteins were identified after two-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectroscopy, including those involved in metabolism, plasmodesmatal remodeling and cell division. The majority of these were not identified by differential RNA expression, demonstrating the importance of proteomics in defining developmental mutants.

During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3’ untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3’ UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3’ UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUS’s O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

Plant growth is controlled by integration of hormonal and light-signaling pathways. BZS1 is a B-box zinc finger protein previously characterized as a negative regulator in the brassinosteroid (BR) signaling pathway and positive regulator in the light-signaling pathway. However, the mechanisms by which BZS1/BBX20 integrates light and hormonal pathways are not fully understood. Here, using a quantitative proteomic workflow, we identified several BZS1-associated proteins including light signaling components COP1 and HY5. Direct interactions of BZS1 with COP1 and HY5 were verified by yeast two-hybrid and co-immunoprecipitation assays. Overexpression of BZS1 causes a dwarf phenotype that is suppressed by the hy5 mutation, while overexpression of BZS1 fused with the SRDX transcription repression domain (BZS1-SRDX) causes a long-hypocotyl phenotype similar to hy5, indicating that BZS1’s function requires HY5. BZS1 positively regulates HY5 expression, whereas HY5 negatively regulates BZS1 protein level, forming a feedback loop that potentially contributes to signaling dynamics. In contrast to BR, strigolactone (SL) increases BZS1 level, whereas the SL responses of hypocotyl elongation, chlorophyll and HY5 accumulation are diminished in the BZS1-SRDX seedlings, indicating that BZS1 is involved in these SL responses. These results demonstrate that BZS1 interacts with HY5 and plays a central role in integrating light and multiple hormone signals for photomorphogenesis in Arabidopsis.