Validación de la propuesta Análisis de los resultados

By eye, the top ten HeLa minus arsenite samples by p-value were encouraging (Table 4.7). The differences between the IgG and UNR samples were quite pronounced. There were two entries associated with UNR. As stated previously, it is unlikely that E9PLTO is physiologically relevant and was likely to have been flagged up as a result of parsimony-based algorithms assigning relatively more peptides to a shorter entry than for a larger one. It was reassuring to see that it is not the only entry in the top ten. Furthermore, three of the top ten hits have been mentioned previously (LDB1, NARR and SSBP3). The last of these was present in the company of the functionally related SSBP2. The top hit, showing more than a tenfold increase in UNR samples over IgG samples is HNRNPR. This, and the ribosomal protein S18, were also in keeping with the assumption that UNR interacts with and modulates the translation of mRNAs. The other two hits, a P2Y receptor and a complement protein are interesting but their relevance were not immediately apparent.

There was some overlap between the top ten putative UNR-interacting proteins in non-arsenite treated HeLa cells by t-test p-value (Table 4.7) and by UNR/IgG ratio (Table 4.9). SSBP2 and SSBP3 were both present, as was NARR, LDB1 and the complement protein C4-A. LMO4 had the highest non-infinite ratio, giving strong support to the idea that this protein is a true UNR-interactor. FUS and the related TAF15 are members of the TET family that has a variety of cellular functions (Takahama et al., 2008). These include the control of alternative splicing and transcription (Ishigakiet al., 2012), the regulation of a variety of different protein levels in neurons (Ibrahim

et al., 2013) and they are involved in various pathologies (discussed in Schwartz et al. 2015). Interestingly, although the HeLa cells in this case were unstressed, both FUS and TAF15 are known to migrate to stress granules in stressed cells (Blechingberg et al., 2012). Another interesting protein in the top ten UNR/IgG ratio list was the ubiquitin E3 ligase HUWE1. HUWE1, which has been linked to multiple cancers, has multiple aliases (succinctly reviewed in Choe et al. 2016). It has been reported to promote the restart of replication at stalled replication forks (Choeet al., 2016). It is also linked to X-linked intellectual impairment (Orivoli et al., 2016). Intriguingly, the Huwe1 gene is host to miR-98 and the expression of the pair was shown to be positively correlated (Xu et al., 2015). Furthermore, miR-98 was shown to downregulate the effector caspase, Caspase 3 (Xu et al., 2015). Fas expression was also shown to be downregulated by miR-98 (Wang et al., 2011). Whilst not directly related to the putative interaction between UNR and HUWE1, per se, these findings regarding miR-98 imply that

HUWE1 gene can have an oncogenic function beyond the HUWE1 protein by reducing the ability of the cell to undergo apoptosis. The top hit by ratio was Interleukin enhancer-binding factor 2. It was not present in the IgG samples and was by far the lowest of the top ten in terms of the amount being bound to UNR. It is also known as NF45 and can reduce the production of mature miRNAs in conjunction with ILF3 (also known as NF90) (Sakamotoet al., 2009). It is upregulated in non-small cell lung cancer, where it is correlated with a poor prognosis (Niet al., 2015), and is involved in splicing and in the DNA damage response in 1q21-amplified multiple myeloma (Marchesini et al., 2014). It was noted that multiple myeloma related proteins had been discussed previously (Figure 4.11).

The top ten putative UNR-interacting proteins from arsenite-treated HeLa cell lysates by t-test p-value were also of interest (Table 4.8). As well as many of the common hits (e.g. SSBP3, NARR, SSBP2 and LMO4), there were five proteins directly linked to RNA. These were the ribosomal proteins S23 and L38, eukaryotic translation initiation factor 2A and two entries for Ribonuclease inhibitor. It was worth noting that the recombinant RNase OUT ribonuclease inhibitor (Invitrogen) had been added to the samples prior to the RIP step. That observation led to two assumptions; either the highlighted RNase inhibitor was the exogenous RNase Out or, alternatively, the human protein that was suggested was genuinely present. Given that the Mascot searches used a human database, either option seems reasonable. As RNase inhibitors could be associated with the RNA species that were part of RNP complexes with UNR, that could imply that UNR was associated with more RNA species, or had at least pulled down more RNA molecules, in the plus arsenite samples than in the minus arsenite samples. The other hit, anaplastic lymphoma receptor tyrosine kinase, is interesting due to it being a membrane-bound protein. It is involved in neuronal differentiation (Gouziet al., 2005) and, unusually, it is activated in a ligand-independent manner (Deuel, 2013). It can become fused to a variety of proteins by genetic translocations, these include MYH9 which had been suggested as the top hit by total ion intensity in Scaffold (Table 4.6) (Lamantet al., 2003).

The top ten putative UNR-interacting proteins in arsenite-treated HeLa cells by UNR/IgG ratio contained many of the proteins already discussed (Table 4.10). These included; TAF15, FUS, SSBP2, LMO4, NARR, UNR itself and UNRIP. The remaining proteins included Ran GTPase- activating protein 1 (RANGAP1). The canonical function of a GAP protein is to activate the GTPase activity of small monomeric G proteins, thereby causing them to cleave their associated GTP and functioning as an ‘off switch’. The rangap1 protein was shown to function in the

regulation of kinetochores and knocking it down prevented activation of the spindle checkpoint (Arnaoutov & Dasso, 2003). SUMOylation with SUMO1 was involved with causing RANGAP1 to migrate to the nuclear pore (Matuniset al., 1998). RANGAP1 has also been associated with a number of cancers including diffuse large B cell lymphoma (Changet al., 2013). The ‘TRK-fused gene’ protein is involved in regulating endoplasmic reticulum structure and protein release (Beetzet al., 2013). It is interesting in that it is a potential fusion partner for one of the proteins noted above as a top hit by t-test from lysate made from arsenite-treated HeLa cells (ALK, Table 4.8A). The last remaining suggested top hit was the same P2Y receptor (P2RY12) as was suggested in the untreated HeLa samples by t-test. It is involved in platelet activation (discussed in Dorsam & Kunapuli 2004) and is the target of clopidogrel (Saviet al., 2001).