The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

Abstract

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.

Keywords: Neurospora crassa; RNA interference (RNAi); cap-binding proteins (CBPs); meiosis; meiotic silencing by unpaired DNA (MSUD).

Figure 1

The absence of CBC correlates with a reduction in MSUD activity. Here, crosses heterozygous for r^∆ were examined. When MSUD is proficient, it silences the unpaired r⁺ gene, which results in the production of mostly round ascospores [with only 2.6% of the progeny being normal (American football-shaped); cross 1]. When both parents lack CBP20 and CBP80, MSUD becomes deficient and considerably more normal ascospores (21.6%) can be observed (cross 4). Crosses homozygous for either single cbp knockout exhibit a more modest MSUD suppression (with 8.5% and 14.2% normal ascospores; crosses 2 and 3, respectively). In comparison, MSUD appears to be completely suppressed in a sad-5-null background (Hammond et al. 2013b). Crosses were performed in triplicate, with an average of ∼100 ascospores/cross examined. +, wild type (WT) at cbp loci. 1, F2-29 × P3-08. 2, F6-20 × P20-47. 3, F6-19 × P20-42. 4, F6-15 × P20-68.

Figure 2

CBC is not essential for the vegetative stage. Mutations in cbp20 and/or cbp80 do not appear to have a substantial effect on the linear growth of the fungus. Strains used in this assay (performed in triplicate) include P3-08, P20-38, P20-47, and P20-68.

Figure 3

CBC-deficient crosses exhibit impaired sexual development. (A) The loss of CBP20 and/or CBP80 is associated with a considerable reduction in ascospore production. (B) Although protoperithecial development appears proficient in a given single or double cbp mutant, there only seem to be roughly half the normal number of mature perithecia in the corresponding homozygous cross. Dissection of mutant perithecia showed that they contain frequent ascus abortions. Deletion of sad-5, in comparison, does not significantly affect ascospore production (Hammond et al. 2013b). 1, F2-01 × P3-08. 2, F8-02 × P20-47. 3, F6-19 × P20-39. 4, F8-04 × P20-68.

Figure 4

CBC localizes predominantly in the nucleus. (A–D) CBP20 and CBP80 colocalize in the nucleus, excluding the nucleolus (arrow). P20-44 × P20-45. (E–H) CBP80 does not appear to linger outside the nuclear envelope (labeled by nucleoporin NUP120). P20-34 × P20-35. (I–L) CBP20 accumulates outside the nuclear envelope in the absence of CBP80 (arrow), suggesting that its robust nuclear entry (and/or reentry) requires the latter. P23-28 × P23-29. The chromatin was stained with DAPI. Bar, 5 µm.

Figure 5

Interactions among CBP20, CBP80, and SMS-2. Similar to their eukaryotic counterparts, Neurospora CBP20 and CBP80 form a complex (A). The two cap-binding proteins interact with the SMS-2 Argonaute (D and E) and not SAD-5 (B and C). In a BiFC assay, each protein is tagged with either the N- or C-terminal fragment of YFP. A positive interaction (F) reconstitutes the yellow fluorophore, while a negative interaction (G) does not. Micrographs illustrate prophase asci expressing (A) yfpn-cbp20 and yfpc-cbp80 (P21-10 × P21-11), (B) yfpn-cbp20 and yfpc-sad-5 (P21-52 × P23-26), (C) yfpn-cbp80 and yfpc-sad-5 (P21-53 × P21-54), (D) yfpn-cbp20 and yfpc-sms-2 (P15-36 × P25-55), (E) yfpn-sms-2 and yfpc-cbp80 (P25-56 × P25-57), (F) yfpn-sms-2 and sad-1-yfpc (P18-19 × P18-21), and (G) yfpn and yfpc (P6-59 × P8-25). The chromatin was stained with DAPI. Bar, 5 µm.