The COpy Number ANalysis (CONAN) database (accessible from the COSMIC website) shows copy number changes recorded in cancer samples. It flagged up a number of copy number changes inUNR. An alignment of these data with similar data for NRASshowed that theUNR data was a subset of theNRASdata (data not shown). AsNRASis a known protooncogene and UNRis not, it was considered likely that the changes in copy number forNRASwould outweigh and confound any equivalent changes inUNRcopy number. For this reason it was assumed to be unworthy of further consideration.
In another investigation, the expression profile of 4 pancreatic cancer cell lines and two pancreatic cancer primary cell lines were taken and compared with 11 libraries of non-neoplastic tissue that included both primary and cultured cells. The comparison showed 15UNRtags per million in non-neoplastic samples and 144UNRtags per million in the pancreatic cancer samples (Hustinx et al., 2004). Confoundingly, the tag they used for UNR was shared with that for lumican, a collagen binding protein reported to be upregulated in breast and colorectal cancer (Lu et al., 2002). As the same sequences were present in each transcript, obviously, they shared the statistics and it is impossible to infer anything about the proportion of relative upregulation accounted for by each transcript.
1.7 UNRIP
UNRIP is a known UNR-interacting protein (Hunt et al., 1999). TheUnripgene is believed to be expressed in all mouse tissues with high levels of expression observed in the liver and testicular tissue (Datta, Chytil, Gorska, & Moses, 1998). In comparison,Unrtranscript levels were reported to be 7 times lower in liver compared to testis (Boussadia et al., 1997). The UNRIP gene is universally conserved among the eukaryotae from yeast to mammals (Datta et al., 1998), is often upregulated in cancer (e.g. in a variety of lung cancers, as compared with healthy lung tissue from the same patients (Halder et al., 2006)), and has been shown to be essential in mice (Chen,
Delrow, Corrin, Frazier, & Soriano, 2004). It is reported to be one of ten oncogenes responsible for the phenotype of one of the cancer cell lines used in this study – the SaOS-2 osteosarcoma cell line (Niforou et al. 2006).
Whilst UNRIP is not believed to possess a nuclear localization signal, it is observed in the nucleus (Halder et al., 2006). One protein with which it can interact is GEMIN7, part of the survival motor neuron (SMN) complex (Grimmler et al., 2005). The SMN complex is involved in splicing and consists of a variety of proteins including the eponymously named SMN protein and GEMINs 2-7 (Gubitz et al., 2004). Unrip was later shown to interact with the SMN complex via GEMIN7, although this relationship was only apparent in the cytoplasm. Knocking downUNRIPincreased the nuclear localisation of the SMN complex (Grimmler et al., 2005). Interestingly, the interaction between Unrip and either GEMIN7 or UNR was noted to be mutually exclusive (Grimmler et al., 2005). Later work showed that the binding of another Unrip binding partner, La-related protein 6 (Larp6), was also mutually exclusive to both Gemin7 and Unr (Vukmirovic, Manojlovic, & Stefanovic, 2013). Protein sequences for UNR (O75534), LARP6 (Q9BRS8) and GEMIN7 (Q9H840) are freely available at www.uniprot.org. Manual comparison of these sequences suggests a shared consensus Unrip binding motif VLRxPRGPD (Figure 1.9). Importantly, a mutually exclusive relationship between UNRIP and either UNR or one of two other proteins implies that UNR function through UNRIP could be competitively reduced by the presence of LARP6 or GEMIN7. Also, UNR could competitively reduce the activity of UNRIP with LARP6 and GEMIN7.
Figure 1.9: Conserved residues in mutually exclusive Unrip binding proteins imply a putative Unrip binding consensus sequence. Alignments were carried out by hand based on the www.uniprot.org human protein sequences O75534 (UNR), Q9BRS8 (LARP6) and Q9H840 (GEMIN7). Yellow shading signifies residue shared by all three proteins. Green shading signifies a serine or a threonine. Black shading signifies that a gap was intentionally inserted into a protein sequence to improve the alignment (subjectively) between the three proteins. N.B. This is similar to a diagram in Vukmirovic et al. (2013) although the amino acid range considered here is greater (their diagram runs from VRL to GF). They align the phenylalanine of GEMIN7 that lies immediately before the green-shaded serine with asparagines from the other two proteins – i.e. they shift the spaces from N-S/T to D-N).
UNRIP was shown to interact with Transforming Growth Factor beta (TGFβ) Receptor 1 and 2 (TGFβR1/2) homodimers in the presence or absence of receptor agonist (Datta & Moses, 2000). It was shown to interact with a variety of Sister of Mothers Against Decapentaplegic (SMAD) proteins; including SMAD2, SMAD3 and SMAD7 (Datta & Moses, 2000). UNRIP binding to SMAD7 increases SMAD7 repression of TGFβ signalling (Datta & Moses, 2000). One protein downregulated by UNRIP activity at TGFβ receptors is p21Cipand knocking down UNRIP increases
p21Cipexpression (Halder et al., 2006). Over-expression of UNRIP in cancer therefore leads to
the hyperphosphorylation of the retinoblastoma protein, reducing its capacity to sequester proliferation-promoting E2F transcription factors, and driving cellular proliferation (Harbour & Dean, 2000).
The 3-phosphoinositide dependent kinase PDK1 binds to protein kinase B (PKB/AKT) and recruits it to the membrane where it can then act as one of the two PKB activating kinases. Other key targets include the ribosomal protein S6 kinase 1 (S6K1) and the atypical protein kinase C zeta (PKCζ) (Berridge, 2014). UNRIP/Unrip interacts with PDK1/Pdk1 and the interaction is enhanced by insulin signalling. The insulin effect is likely to bePI-3,4,5-P3-dependent asit was not observed when cells were treated with wortmannin (Seong, Jung, Choi, Kim, & Ha, 2005). This indirect link between insulin signalling and UNR via UNRIP is worth considering in light of work linking UNR expression to control of diabetes. That work showed by qPCR that theUNRtranscript level went up over 6 fold after one week of treatment in patients who were admitted to hospital with poorly controlled diabetes (Xavier et al., 2014). Interestingly, in light of current findings in this work linking UNR to selenium compound metabolism (see section 4.11.6), there were two other genes that were considered significant by the authors; one of which being Selenoprotein S, which fell more than three-fold following treatment (Xavier et al., 2014).
UNRIP has been shown to interact with the pro-apoptotic apoptosis signal regulating kinase 1 (ASK1), an interaction promoted by ASK1 phosphorylating UNRIP at T175 and S179. The addition of UNRIP was shown to decrease the amount of hydrogen peroxide-instigated apoptosis, whereas knocking down UNRIP increased the proportion of cells undergoing apoptosis in response to hydrogen peroxide (Jung, Seong, Manoharan, & Ha, 2010).
Drawing together the previous two sections, the pro-survival PDK1 was shown to interact with the pro-apoptotic ASK1 and that mutual phosphorylation reduced their respective downstream signalling activities (Seong, Jung, Ichijo, & Ha, 2010).
Unrip interacts with the Ewing’s Sarcoma protein (EWS) in the nucleus, inhibiting an alternative interaction with the transcriptional co-activator EP300/CBP and downregulating EWS target genes such asc-fos(Anumanthan, Halder, Friedman, & Datta, 2006). As stated previously, UNR interacts with other proteins on the mCRD of the c-fos transcript, both stabilising and promoting degradation of the message following translation. EP300/CBP is required for EWS to activate hepatocyte nuclear factor 4 (HNF4)-mediated transcription (Araya et al., 2003). HNF4-mediated transcription is important in differentiation. For example, dedifferentiated hepatoma cells can be forced to re-differentiate following the stable expression of Hnf4 (Späth & Weiss, 1998). Gata6 is involved in transcribing theHnf4gene within primitive endoderm and this is required for differentiation into visceral extraembryonic endoderm (Morrisey et al., 1998). As discussed previously, the essential function of Unr in mice was linked to destabilisation of the Gata6 transcript (Elatmani et al., 2011).
UNRIP has been shown to interact with nuclear export factors and has also been suggested to be part of neuronal transport granules that transfer mRNAs into the cytoplasm and around the cell linked to microtubules (Tretyakova et al., 2005).
There are many other reported roles for UNRIP in the literature that shall be overlooked here for the sake of brevity. Based upon the lines of investigation that have been considered here, however, it appears that the functional roles of UNR and UNRIP overlap beyond any physical interactions between the two.