The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription.

The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy.

Macroautophagy/autophagy is a conserved process in eukaryotic cells that mediates the degradation and recycling of intracellular substrates. Proteins encoded by autophagy-related (ATG) genes are essentially involved in the autophagy process and must be tightly regulated in response to various circumstances, such as nutrient-rich and starvation conditions. However, crucial transcriptional activators of ATG genes have remained obscure. Here, we identify the RNA polymerase II subunit Rpb9 as an essential regulator of autophagy by a high-throughput screen of a Saccharomyces cerevisiae gene knockout library. Rpb9 plays a crucial and specific role in upregulating ATG1 transcription, and its deficiency decreases autophagic activities. Rpb9 promotes ATG1 transcription by binding to its promoter region, which is mediated by Gcn4. Furthermore, the function of Rpb9 in autophagy and its regulation of ATG1/ULK1 transcription are conserved in mammalian cells. Together, our results indicate that Rpb9 specifically activates ATG1 transcription and thus positively regulates the autophagy process.

Absence of the Rpb9 subunit of RNA polymerase II reduces the chronological life span in fission yeast.

Fission yeast RNA polymerase II consists of 12 subunits, Rpb1-Rpb12. Among these subunits, Rpb9 is the only subunit whose absence does not cause lethality under optimum growth conditions in fission yeast. However, an rpb9 null fission yeast mutant exhibits a slow-growth phenotype under optimum growth conditions and a defect in survival under environmental and genotoxic stress conditions. To further gain an understanding of its physiological roles, in the present study we have elucidated the role of the Rpb9 subunit in chronological aging using fission yeast as the model organism. Our results provide evidence that the absence of Rpb9 reduces the chronological life span in fission yeast. Our data further shows that lack of Rpb9 in fission yeast causes oxidative stress sensitivity and accumulation of reactive oxygen species during the stationary phase. Our domain mapping experiments have demonstrated that the Rpb9 region encompassing its amino-terminal zinc finger domain and the central linker region is important for the role of Rpb9 in chronological aging. Finally, we also show that expression of the budding yeast or human Rpb9 ortholog can functionally complement the reduced chronological life span phenotype of the fission yeast rpb9 deletion mutant. Taken together, our study has identified a new role of the Rpb9 subunit in chronological aging.

RNA Polymerase II Trigger Loop Mobility: INDIRECT EFFECTS OF Rpb9.

Rpb9 is a conserved RNA polymerase II (pol II) subunit, the absence of which confers alterations to pol II enzymatic properties and transcription fidelity. It has been suggested previously that Rpb9 affects mobility of the trigger loop (TL), a structural element of Rpb1 that moves in and out of the active site with each elongation cycle. However, a biochemical mechanism for this effect has not been defined. We find that the mushroom toxin α-amanitin, which inhibits TL mobility, suppresses the effect of Rpb9 on NTP misincorporation, consistent with a role for Rpb9 in this process. Furthermore, we have identified missense alleles of RPB9 in yeast that suppress the severe growth defect caused by rpb1-G730D, a substitution within Rpb1 α-helix 21 (α21). These alleles suggest a model in which Rpb9 indirectly affects TL mobility by anchoring the position of α21, with which the TL directly interacts during opening and closing. Amino acid substitutions in Rpb9 or Rpb1 that disrupt proposed anchoring interactions resulted in phenotypes shared by rpb9Δ strains, including increased elongation rate in vitro Combinations of rpb9Δ with the fast rpb1 alleles that we identified did not result in significantly faster in vitro misincorporation rates than those resulting from rpb9Δ alone, and this epistasis is consistent with the idea that defects caused by the rpb1 alleles are related mechanistically to the defects caused by rpb9Δ. We conclude that Rpb9 supports intra-pol II interactions that modulate TL function and thus pol II enzymatic properties.

Identification of host proteins interacting with the E protein of porcine epidemic diarrhea virus.

Introduction: Porcine epidemic diarrhea (PED) is an acute, highly contagious, and high-mortality enterophilic infectious disease caused by the porcine epidemic diarrhea virus (PEDV). PEDV is globally endemic and causes substantial economic losses in the swine industry. The PEDV E protein is the smallest structural protein with high expression levels that interacts with the M protein and participates in virus assembly. However, how the host proteins interact with E proteins in PEDV replication remains unknown. Methods: We identified host proteins that interact with the PEDV E protein using a combination of PEDV E protein-labeled antibody co-immunoprecipitation and tandem liquid-chromatography mass-spectroscopy (LC-MS/MS). Results: Bioinformatical analysis showed that in eukaryotes, ribosome biogenesis, RNA transport, and amino acid biosynthesis represent the three main pathways that are associated with the E protein. The interaction between the E protein and isocitrate dehydrogenase [NAD] ß-subunit (NAD-IDH-ß), DNA-directed RNA polymerase II subunit RPB9, and mRNA-associated protein MRNP 41 was validated using co-immunoprecipitation and confocal assays. NAD-IDH-ß overexpression significantly inhibited viral replication. Discussion: The antiviral effect of NAD-IDH-ß suggesting that the E protein may regulate host metabolism by interacting with NAD-IDH-ß, thereby reducing the available energy for viral replication. Elucidating the interaction between the PEDV E protein and host proteins may clarify its role in viral replication. These results provide a theoretical basis for the study of PEDV infection mechanism and antiviral targets.

Transcriptional regulation of autophagy by RNA polymerase II.

Macroautophagy/autophagy is a catabolic recycling pathway and is tightly regulated by upstream signals. Autophagy genes are quickly upregulated upon stimuli such as nutrition limitation in response to the external environment. However, how the transcriptional activation of autophagy genes occurs is not well understood. We recently found that in yeast, the RNA polymerase II subunit Rpb9 specifically and efficiently upregulates the transcription of the autophagy gene ATG1 with the mediation of Gcn4. Such regulation was shown to be essential for autophagic activities induced by starvation. Furthermore, the function of Rpb9 in autophagy and the activation of ATG1 transcription is conserved in mammalian cells. In conclusion, Rpb9 specifically and positively regulates ATG1 transcription as a key regulator of autophagy.

Deletion of the non-essential Rpb9 subunit of RNA polymerase II results in pleiotropic phenotypes in Schizosaccharomyces pombe.

Schizosaccharomyces pombe RNA polymerase II comprises twelve different subunits. Its Rpb9 subunit comprises 113 amino acids, and is the only non-essential subunit of S. pombe RNA polymerase II. However, its functions have not been studied in S. pombe. The results presented in this study demonstrate that Rpb9 is involved in regulating growth under optimum and certain stress conditions in S. pombe. To further address the role (s) of various domains of this subunit in regulating these phenotypes, deletion mutant analysis was done. We observed that the region spanning 1-74 amino acids, encompassing the amino-terminal zinc finger domain and the linker region of Rpb9 was able to rescue the phenotypes associated with rpb9+deletion. We also demonstrate that the functions of this subunit are only partially conserved among yeast and humans. Our computational biology approaches provide a structural basis for the differential role of various Rpb9 domains in S. pombe. Furthermore, using these tools we show that there has been a co-evolution of the interaction residues between the Rpb9 subunit and the two largest subunits of RNA polymerase II, allowing for a more stringent organism-specific packing. Taken together, our results have provided functional and structural insights into the Rpb9 subunit of S. pombe.