Table 1. Many proteins that copurify with LIN-41 associate with OMA-1 or regulate mRNA translation, cytoplasmic polyadenylation, or deadenylation.
| Proteina | Protein coverage (%)b | |||
|---|---|---|---|---|
| Experiment I | Experiment II | Experiment III | Experiment IV | |
| Tandem IP from glycerol-gradient fractions | Tandem IP from high-speed supernatant | GFP IP from high-speed supernatant | GFP IP from high-speed supernatant with RNase Ac | |
| LIN-41 | 48.2 | 40.4 | 51.8 | 48.0 (0.9) |
| mRNA translation | ||||
| MEX-1d | 43.5 | 48.0 | 59.9 | 62.6 (1.0) |
| MEX-3d | 29.1 | 40.9 | 55.8 | 66.8 (1.2) |
| GLD-1d | 41.3 | 39.7 | 45.8 | 43.4 (0.9) |
| MEX-5d | 17.5 | 30.6 | 25.0 | 22.9 (0.9) |
| TIAR-1d | 11.5 | 28.9 | 28.9 | 24.5 (0.8) |
| OMA-1d | 17.2 | 26.5 | 42.0 | 36.4 (0.9) |
| IFE-3 | 17.1 | 25.1 | 43.0 | 43.0 (1.0) |
| PUF-11d | 12.5 | 22.8 | 27.5 | 25.9 (0.9) |
| IFG-1d | 1.9 | 22.2 | 32.6 | 24.5 (0.8) |
| SPN-4d | 2.9 | 21.1 | 36.8 | 34.8 (0.9) |
| OMA-2d | 17.6 | 20.6 | 36.1 | 33.3 (0.9) |
| MEG-1 | 8.5 | 17.6 | 32.1 | 6.8 (0.2) |
| POS-1d | 7.2 | 11.0 | 39.8 | 13.3 (0.3) |
| VBH-1 | 2.6 | 10.8 | 20.6 | 11.1 (0.5) |
| PUF-6 | 2.6 | 10.5 | 12.2 | 5.0 (0.4) |
| NHL-2d | 4.8 | 10.5 | 17.6 | 11.5 (0.7) |
| FBF-1/FBF-2e | 2.3 | 9.6 | 17.9 | 6.2 (0.3) |
| Cytoplasmic polyadenylation | ||||
| GLD-2d | 21.1 | 34.2 | 35.9 | 32.2 (0.9) |
| GLD-3d | 23.2 | 33.2 | 50.2 | 47.2 (0.9) |
| RNP-8 | 6.5 | 26.2 | 43.1 | 32.4 (0.8) |
| Deadenylationf | ||||
| LET-711d | 19.9 | 31.2 | 32.3 | 25.6 (0.8) |
| TAG-153d | 5.5 | 22.1 | 37.0 | 27.8 (0.8) |
| NTL-4d | 22.4 | 20.3 | 38.9 | 30.8 (0.8) |
| NTL-9d | 7.8 | 17.5 | 32.7 | 23.7 (0.7) |
| CCF-1 | 10.0 | 16.1 | 39.0 | 35.2 (0.9) |
| NTL-3 | 4.7 | 13.7 | 18.5 | 17.1 (0.9) |
| NTL-11 | 7.3 | 11.7 | 16.9 | 18.9 (1.1) |
| CCR-4d | 9.9 | 8.3 | 23.4 | 16.8 (0.7) |
| NTL-2 | 4.7 | 4.7 | 28.8 | 21.6 (0.8) |
| Other | ||||
| NCL-1d | 8.1 | 20.8 | 31.1 | 25.6 (0.8) |
| D1081.7 | 3.7 | 20.5 | 36.1 | 31.2 (0.9) |
| CLIK-1 | 61.6 | 19.7 | 33.6 | 3.5 (0.1) |
| LST-3/CCAR-1 | 13.2 | 16.5 | 27.9 | 23.5 (0.8) |
| C05C10.2d | 3.7 | 16.5 | 16.7 | 10.8 (0.6) |
| RLE-1 | 6.8 | 15.6 | 23.2 | 20.2 (0.9) |
| C30G12.2 | 12.1 | 15.5 | 11.3 | 5.7 (0.5) |
| C35A5.8 | 5.4 | 15.5 | 17.4 | 14.3 (0.8) |
| ADR-1d | 9.9 | 15.2 | 17.7 | 17.1 (1.0) |
| LIN-66 | 3.8 | 14.7 | 26.6 | 14.4 (0.5) |
| GLS-1d | 8.3 | 13.6 | 13.6 | 12.1 (0.9) |
| PQN-87d | 2.2 | 13.4 | 18.8 | 14.3 (0.8) |
| NMY-1 | 5.4 | 13.1 | 32.1 | 12.0 (0.4) |
| ERGO-1g | 3.0 | 12.7 | 18.5 | 7.4 (0.4) |
| KIN-2 | 9.3 | 12.0 | 35.3 | 20.2 (0.6) |
| NASP-2d | 5.0 | 11.9 | 35.2 | 42.3 (1.2) |
| H05L14.2 | 1.0 | 10.7 | 16.7 | 10.1 (0.6) |
| EGG-4/EGG-5e | 2.0 | 9.8 | 16.3 | 11.4 (0.7) |
| AIN-2 | 2.7 | 9.6 | 27.2 | 23.9 (0.9) |
IP, immunopurification.
Proteins previously reported to copurify with OMA-1 in the presence of RNase A (see Table 1 and File S2 from Spike et al. 2014b) are indicated in bold font. Proteins reported to directly interact with OMA-1 (Spike et al. 2014b) are underlined. mRNAs encoding the proteins indicated in italic font are enriched fourfold in LIN-41 IPs compared to the input lysate control (see File S3 and below). GLD-1 is reported in Table 1 despite its representation in the control SACY-1 tandem affinity purification because of its interactions with OMA-1 in yeast two-hybrid assays (Spike et al. 2014b). The copurification of GLD-1 and SACY-1 might reflect their involvement in germline sex determination (Francis et al. 1995; Kim et al. 2012). Similarly, IFG-1 and LET-711 are reported despite their representation in the control SACY-1 tandem affinity purification because they copurify with OMA-1 in the presence of RNase A. NTL-9 is reported despite its presence in the SACY-1 control purification because the results indicate that most other components of the CCR4-NOT deadenylase complex copurify with LIN-41.
Peptide coverage in a single gel slice assessed by mass spectrometry in the various LIN-41 purifications from DG3923 lin-41(tn1541[gfp::tev::s::lin-41]) fog-1(q253) extracts. Only proteins showing at least 10% coverage in the tandem IP from the high-speed supernatant are shown, with the exception that all CCR4-NOT coverage data are reported because the data indicate the complex copurifies with LIN-41. The complete data on which Table 1 is based are presented in File S2.
The ratio of the peptide coverage with and without RNase A digestion (experiment IV/experiment III) is indicated in parentheses.
These proteins were detected in a tandem IP from DG3923 high-speed supernatant using a limiting amount of extract (File S2, experiment XIII).
The mass spectrometry data do not differentiate between the indicated paralogs.
Components of the CCR4-NOT deadenylase complex (Nousch et al. 2013) that copurify with LIN-41. tag-153 is a paralog of ntl-2 (http://www.wormbase.org).
In addition to the Argonaute protein ERGO-1 (Gent et al. 2010; Vasale et al. 2010), WAGO-1 and CSR-1 were represented in the LIN-41 tandem affinity purification from the high-speed supernatant. The representation of WAGO-1 (9.1% coverage) was just below the 10% cutoff threshold and CSR-1 (14.8% coverage in the LIN-41 tandem IP) was also represented (7.4% coverage) in the SACY-1 control IP. Previously, Duchaine et al. (2006) reported the recovery of LIN-41 in DCR-1 IPs (7% coverage). In our tandem affinity LIN-41 IPs DCR-1 exhibited 1% coverage.