Heterodimerization of Munc13 C2A domain with RIM regulates synaptic vesicle docking and priming

The presynaptic active zone protein Munc13 is essential for neurotransmitter release, playing key roles in vesicle docking and priming. Mechanistically, it is thought that the C2A domain of Munc13 inhibits the priming function by homodimerization, and that RIM disrupts the autoinhibitory homodimerization forming monomeric priming-competent Munc13. However, it is unclear whether the C2A domain mediates other Munc13 functions in addition to this inactivation–activation switch. Here, we utilize mutations that modulate the homodimerization and heterodimerization states to define additional roles of the Munc13 C2A domain. Using electron microscopy and electrophysiology in hippocampal cultures, we show that the C2A domain is critical for additional steps of vesicular release, including vesicle docking. Optimal vesicle docking and priming is only possible when Munc13 heterodimerizes with RIM via its C2A domain. Beyond being a switching module, our data suggest that the Munc13-RIM heterodimer is an active component of the vesicle docking, priming and release complex

The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production

qiRNA is a new type of small interfering RNA induced by DNA damage

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNA) and microRNAs, several types of endogenously produced small RNAs play important roles in gene regulation, germ cell maintenance and transposon silencing 1–4. Production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases (RdRPs) to make double stranded RNA (dsRNA). The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a novel class of small RNAs in the filamentous fungus Neurospora. This class of small RNAs, named qiRNAs for their association with QDE-2, are about 20–21 nt long (several nt shorter than Neurospora siRNAs) with a strong preference for uridine at the 5′ end and originate mostly from the ribosomal DNA locus. Production of qiRNAs requires the RdRP QDE-1, the Werner/Bloom RecQ DNA helicase homolog QDE-3 and dicers. qiRNA biogenesis also requires DNA damage-induced aRNAs as precursor, a process that is dependent on QDE-1 and QDE-3. Surprisingly, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. In addition, the Neurospora RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs in DNA damage response by inhibiting protein translation

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn't inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs

NDAT Targets PI3K-Mediated PD-L1 Upregulation to Reduce Proliferation in Gefitinib-Resistant Colorectal Cancer

The property of drug-resistance may attenuate clinical therapy in cancer cells, such as chemoresistance to gefitinib in colon cancer cells. In previous studies, overexpression of PD-L1 causes proliferation and metastasis in cancer cells; therefore, the PD-L1 pathway allows tumor cells to exert an adaptive resistance mechanism in vivo. Nano-diamino-tetrac (NDAT) has been shown to enhance the anti-proliferative effect induced by first-line chemotherapy in various types of cancer, including colorectal cancer (CRC). In this work, we attempted to explore whether NDAT could enhance the anti-proliferative effect of gefitinib in CRC and clarified the mechanism of their interaction. The MTT assay was utilized to detect a reduction in cell proliferation in four primary culture tumor cells treated with gefitinib or NDAT. The gene expression of PD-L1 and other tumor growth-related molecules were quantified by quantitative polymerase chain reaction (qPCR). Furthermore, the identification of PI3K and PD-L1 in treated CRC cells were detected by western blotting analysis. PD-L1 presentation in HCT116 xenograft tumors was characterized by specialized immunohistochemistry (IHC) and the hematoxylin and eosin stain (H&E stain). The correlations between the change in PD-L1 expression and tumorigenic characteristics were also analyzed. (3) The PD-L1 was highly expressed in Colo_160224 rather than in the other three primary CRC cells and HCT-116 cells. Moreover, the PD-L1 expression was decreased by gefitinib (1 µM and 10 µM) in two cells (Colo_150624 and 160426), but 10 µM gefitinib stimulated PD-L1 expression in gefitinib-resistant primary CRC Colo_160224 cells. Inactivated PI3K reduced PD-L1 expression and proliferation in CRC Colo_160224 cells. Gefitinib didn’t inhibit PD-L1 expression and PI3K activation in gefitinib-resistant Colo_160224 cells. However, NDAT inhibited PI3K activation as well as PD-L1 accumulation in gefitinib-resistant Colo_160224 cells. The combined treatment of NDAT and gefitinib inhibited pPI3K and PD-L1 expression and cell proliferation. Additionally, NDAT reduced PD-L1 accumulation and tumor growth in the HCT116 (K-RAS mutant) xenograft experiment. (4) Gefitinib might suppress PD-L1 expression but did not inhibit proliferation through PI3K in gefitinib-resistant primary CRC cells. However, NDAT not only down-regulated PD-L1 expression via blocking PI3K activation but also inhibited cell proliferation in gefitinib-resistant CRCs

qiRNA is a new type of small interfering RNA induced by DNA damage

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNA) and microRNAs, several types of endogenously produced small RNAs play important roles in gene regulation, germ cell maintenance and transposon silencing 1–4. Production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases (RdRPs) to make double stranded RNA (dsRNA). The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a novel class of small RNAs in the filamentous fungus Neurospora. This class of small RNAs, named qiRNAs for their association with QDE-2, are about 20–21 nt long (several nt shorter than Neurospora siRNAs) with a strong preference for uridine at the 5′ end and originate mostly from the ribosomal DNA locus. Production of qiRNAs requires the RdRP QDE-1, the Werner/Bloom RecQ DNA helicase homolog QDE-3 and dicers. qiRNA biogenesis also requires DNA damage-induced aRNAs as precursor, a process that is dependent on QDE-1 and QDE-3. Surprisingly, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. In addition, the Neurospora RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs in DNA damage response by inhibiting protein translation

Control of WHITE COLLAR localization by phosphorylation is a critical step in the circadian negative feedback process

Reversible protein phosphorylation has critical functions in the eukaryotic circadian negative feedback loops. In Neurospora, the FREQUENCY protein closes the circadian negative feedback loop by promoting the phosphorylation of its transcription activator, the WHITE COLLAR complex (WCC) and consequently inhibiting WCC activity. Here we show that protein phosphatase 4 is a novel component of the Neurospora clock by regulating both processes of the circadian negative feedback loop. The disruption of pp4 results in short period rhythms with low amplitude. In addition to its role in regulating FRQ phosphorylation and stability, PP4 also dephosphorylates and activates WCC. In contrast to PP2A, another phosphatase that activates WCC, PP4 has a major function in promoting nuclear entry of WCC. PKA, a WC kinase, inhibits WC nuclear localization. Furthermore, the FRQ-dependent WC phosphorylation promotes WCC cytosolic localization. Together, these results revealed WCC nucleocytoplasmic shuttling as an important step in the circadian negative feedback process and delineated the FRQ-dependent WCC inhibition as a two-step process: the inhibition of WCC DNA-binding activity followed by sequestration of WCC into the cytoplasm