In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD), which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi) proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC). Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors.
Although detection of hepatitis C virus RNA with polymerase chain reaction has become the standard for diagnosis, extensive application has been thwarted by polymerase chain reaction's labor intensiveness, risk of false-positive results through contamination and time required for individual assays. To minimize these limitations, we developed and validated a one-step hepatitis C virus RNA polymerase chain reaction assay. The one-step method was compared with traditional hepatitis C virus RNA polymerase chain reaction using primers from the highly conserved 5' untranslated region of the hepatitis C virus genome. Variables studied in the one-step method included the source and quantity of reverse transcriptase (RTase), the concentration of MgCl2 and the duration of reverse transcription and complementary DNA amplification cycles. Optimal conditions for the one-step method were obtained with 25 U of reverse transcriptase and 2 mmol/L MgCl2. The one-step method substantially reduced the time required for analysis. The sensitivity of the one-step method was comparable to that of traditional hepatitis C virus RNA polymerase chain reaction using serially diluted RNA extracted from the serum of a hepatitis C virus-infected patient. The specificity of the one-step method was confirmed on Southern-blot hybridization. The results exhibited 100% concordance with results of traditional hepatitis C virus RNA polymerase chain reaction in 50 serum samples, including those of positive and negative controls. In addition, 100% concordance was observed between the two methods' results when sera containing low levels of hepatitis C virus RNA were used.(ABSTRACT TRUNCATED AT 250 WORDS)
In Neurospora crassa, expression from an unpaired gene is suppressed by a mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD utilizes common RNA interference (RNAi) factors to silence target mRNAs. Here, we report that Neurospora CAR-1 and CGH-1, homologs of two Caenorhabditis elegans RNA granule components, are involved in MSUD. These fungal proteins are found in the perinuclear region and P-bodies, much like their worm counterparts. They interact with components of the meiotic silencing complex (MSC), including the SMS-2 Argonaute. This is the first time MSUD has been linked to RNA granule proteins.
During the sexual phase of Neurospora crassa, unpaired genes are subject to a silencing mechanism known as meiotic silencing by unpaired DNA (MSUD). MSUD targets the transcripts of an unpaired gene and utilizes typical RNA interference (RNAi) factors for its process. Using a reverse genetic screen, we have identified a meiotic silencing gene called sad-9, which encodes a DEAD-box RNA helicase. While not essential for vegetative growth, SAD-9 plays a crucial role in both sexual development and MSUD. Our results suggest that SAD-9, with the help of the SAD-2 scaffold protein, recruits the SMS-2 Argonaute to the perinuclear region, the center of MSUD activity.
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