Interactions of CAF1-NOT complex components from Trypanosoma brucei

Yeast two-hybrid screens

Our trypanosome yeast two-hybrid prey library was made by random shotgun genomic cloning. The library has several million independent yeast clones, each of which expresses a different protein fragment (with roughly 1/12 being within an open reading frame and in-frame)¹⁵. NOT2, NOT10, NOT11 and CAF40 were used as baits to screen the library by mating. We also included another protein of interest, CFB1. CFB1 is expressed in bloodstream-form trypanosomes. Its function is somewhat enigmatic, though it is probably required for optimal cell growth¹⁶. All results are in Table S1. For the NOT complex components, at least one million diploid progeny were subjected to selection, resulting in between 100 and 800 surviving colonies, from which inserts were amplified and subjected to high-throughput sequencing. To find interacting proteins, we selected open reading frames for which there were at least two different in-frame sequences represented by at least 20 reads (Table S1, sheet 2). This procedure gives false positives and false negatives. Poor folding could result in a false positive due to aberrant exposure of hydrophobic surfaces, or in a false negative due to incorrect formation of a folding-dependent interaction domain. In addition, proteins for which the whole protein, or a (near-) complete N-terminus, are required for interaction would be excluded.

We obtained 6, 158, 15 and 3 interaction partners for NOT2, NOT10, NOT11 and CAF40 respectively; CFB1 interacted promiscuously with over 800 partners. (These did not include the only validated partner, MKT1, but we had previously found that MKT1 seems only to interact as a complete protein¹⁵.) To judge the likely specificity of the interactions, we compared them with those of MKT1¹⁵, RBP10 (Mugo and Clayton; unpublished study, manuscript submitted), 4EIP and Tb927.7.2780 (unpublished studies; Terrao, ZMBH). The result for NOT10 strongly suggested a tendency for non-specific interactions, although not as severe as for CFB1. The most specific interactions for NOT2, NOT10 and NOT11 are shown in Table 1. It was notable that no interactions with either CAF1 or NOT1 were detected; this might be because the fragments encoded in our prey library are too small. NOT11 had two unique interactions, with a DNAj domain protein (Tb927.9.1560) and mitochondrial EF-Tu; the latter is unlikely to be physiological because of the protein location. There were also two interactors that were shared only with the promiscuous CFB1 - a protein phosphatase and a protein of unknown function, both of which are probably in the cytoplasm. NOT11 interacted with itself, and shared 7 other interactions with NOT10. Some of these, such as the interaction between NOT10 and NOT11, are likely to be genuine - NOT10 interacts with NOT11 in yeasts^4,17. Apart from NOT11, none of these proteins interacts with bloodstream-form trypanosome mRNA¹⁴ or has any effect in the tethering assay^14,18.

To supplement results from the random fragment library, we used recombinant CAF40 to screen a cDNA protein expression array and a similar array displaying proteins from full-length open reading frames of proteins involved in mRNA metabolism^14,19. Two other proteins, Aurora kinase B and T. brucei polo-like kinase, were included as bait controls to exclude "sticky" proteins. Using the open reading frame array, no specific interaction partners were found. For the cDNA array, selected clones were sequenced, then checked in a pairwise yeast-two hybrid assay. The only confirmed interaction was with the nascent polypeptide associated complex alpha subunit-like protein (Tb927.9.8100/8130). An interaction between the yeast NOT complex and the nascent polypeptide associated complex was previously reported in yeast; the beta subunit Egd1p, had a weak two-hybrid interaction with Caf40p, but much stronger interactions were detected between Egd1p and some other NOT complex components²⁰. The significance of the possible interaction is unclear since the nascent polypeptide associated complex was not found in the affinity purification (see below).

Proteins that co-purify with V5-tagged CAF40

To find proteins that co-purify with CAF40, we integrated a sequence encoding a V5 tag in frame with the 5' end of the open reading frame in the genome of bloodstream form trypanosomes²¹. The resulting parasites are expected to express N-terminally V5-tagged CAF40 at an approximately normal level. Eluates from two independent pull-downs - one with RNase inhibitor, and the other with RNase - were analysed by mass spectrometry, in parallel with a single control from wild-type cells (no tag) (Table S2, sheet 1). As additional controls we used the results from three other tandem affinity purifications of tagged GFP. To detect proteins that are frequent contaminants after affinity purification we compared the results with those from many other experiments from our and other labs. Since our CAF40 experiment included only two replicates, all of the results need further confirmation before definitive conclusions can be drawn. Nevertheless, V5-tagged CAF40 showed specific, reproducible RNA-independent pull down of the NOT1, NOT10, NOT11 and CAF1 subunits of the CAF1-NOT complex (Table S2, sheet 2), while co-purification of NOT2 and NOT5 was diminished by RNAse treatment (Table S2, sheet 3). The results also suggested RNA-independent association with three potential RNA-binding proteins, ZC3H8, ZC3H30 and ZC3H46. Interestingly, all three of these RNA-binding proteins were found associated with mRNA¹⁴ and each one repressed expression in tethering screens^14,18. We therefore speculate that they may repress expression by recruiting the NOT complex via CAF40. Some of the proteins that were pulled down in the absence of RNase also appeared interesting; they included 7 known or potential RNA-binding proteins (CSBII, DRBD4 (PTB2), PUF6, ZC3H28 and ZC3H41, HNRNPH and Tb927.11.14090), and the 5'-3' exoribonuclease XRNA (at rather low coverage). This was, however, only a single experiment, so the results will not be discussed further.

Yeast two-hybrid analysis

The Matchmaker GAL4 Two-Hybrid System3 (Clontech) was used, with Gateway cloning of amplified open reading frames to create bait plasmids. The trypanosome prey library, sequencing methods and bioinformatic analysis have been previously described¹⁵. Briefly, bait plasmids were transformed into AH109 yeast, and the pool of prey plasmids were transformed into the Y187 strains. The cells were allowed to mate, plated on SD agar plates lacking tryptophan, leucine, or both (double drop-out medium) to calculate mating efficiency, then plated without tryptophan, leucine, adenine and histidine (quadruple drop-out medium), and incubated for 3 to 5 days at 30°C. The resulting colonies were re-plated on quadruple drop-out SD plates with 40 μg/ml X-α-Gal and 3-amino triazole (3-AT, 0.5 to 2mM), and incubated for 3 to 5 days. Blue colonies were then pooled and grown overnight in quadruple drop-out medium with 3-amino triazole for plasmid isolation. The plasmid inserts were amplified with bar-coded primers, pooled and sent for library preparation by David Ibbersson (BioQuant, Heidelberg, Germany), then sequenced (Illumina Hi-Seq, EMBL, Heidelberg., Germany). The numbers of reads obtained are in Table S1, Sheet 1.

Reads were aligned to the T. brucei 927 genome, as described¹⁵. We selected sequences that were present at least 10 times, and that had annotated open reading frames that were in frame with the DNA-binding domain (Table S1, sheets 3–5). We then chose a list of unique open reading frames with only one copy each of repeated genes²². We also restricted the selection to open reading frames that included no more than 13 codons upstream of the start codon, and included at least 6 codons before the stop codon. Individual results are in Table S1, sheets 4–7 and final results are summarized in Table S1, sheet 2. Sheet 3 includes locations found in screens with more than three different baits.

Affinity purification of V5-CAF40 and mass spectrometry

About 10¹⁰ bloodstream form cultured bloodstream-form T brucei carrying endogenously V5-tagged CAF40 were subjected to immunoprecipitation, as previously described¹³. The eluate was run 1 cm in 10% SDS PAGE gel, and was cut in 6 pieces for Mass-Spectrometry analysis. The piece were transferred to a 96-well plate and reduced, alkylated and digested with trypsin, as described²³, except that triethylammoniumbicarbonate buffer was used instead of ammoniumbicarbonate buffer. Following digestion, tryptic peptides were extracted from the gel pieces with 50% acetonitrile/0.1% TFA, and concentrated nearly to dryness in a speedVac vacuum centrifuge. Peptides were separated on an analytical column (75um × 150mm) with a flow rate of 300nl/min (nanoAcquity, Waters) using 1% formic acid and an acetonitrile gradient increasing from 3% acetonitrile to 37% acetonitrile over 50 minutes. The UPLC system was on-line coupled to an ESI LTQ Orbitrap XL MS (Thermo Fisher). One survey scan (res: 60000) was followed by 5 information dependent product ion scans in the LTQ. Only doubly and triply charged ions were selected for fragmentation.

Data were analyzed using Proteome Discoverer 1.4 and Mascot (Matrix Science; version 2.4). Mascot was set up to search the the TriTrypDB_9.0_Tbrucei_2015Jan database (20400 entries) using trypsin as protease, a fragment ion mass tolerance of 0.50 Da and a parent mass tolerance of 100 ppm. Iodoacetamide derivative of cysteine was specified in Mascot as a fixed modification. Deamidation of asparagine and glutamic acid, as well as oxidation of methionine, were specified in Mascot as variable modifications. Target decoy PSM validator was set to a target FDR (strict) of 0.01. Results are in Table S2.