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The PAF1 complex binds RNA polymerase, is highly conserved among eukaryotes and plays roles in human disease such as cancer. The PAF1 complex affects the amount of numerous RNAs by affecting transcriptional and post-transcriptional

processes (Penheiter et al. 2005; Tomson and Arndt 2013). Here, it was investigated how the PAF1 complex affects fitness of yeast cells with defective telomeres to help explore the potential relevance of these interactions to cancer.

The experiments in this Chapter show that the PAF1 complex has numerous effects on telomere biology and the data is summarized by Figure 5-12. A new set of

interactions between the PAF1 complex and components of the ESCRT machinery have also been described. For instance, I found that Paf1 and Ctr9 are essential for viability in cells with defects in ESCRT-I, ESCRT-II or ESCRT-III complexes. All the data here described, and much of the literature, support the view that Ctr9 and Paf1 are the most critical members of the PAF1 complex. Conversely, Leo1 and Rtf1 are the least critical (auxiliary) members of the PAF1 complex, and Cdc73 is somewhere in-between.

Consistent with previous reports, the results here described find that the PAF1

complex is needed to achieve normal, high levels of TLC1 (telomerase component)

RNA (Mozdy et al. 2008). Leo1 and Rtf1 have lesser effects than Cdc73, Ctr9 and

Paf1 on TLC1 levels (Pathway a, Figure 5-12). Also, I show for the first time that all

the PAF1 complex components induce VPS36 transcript levels, with a stronger role

for Paf1 and Ctr9 (Pathway b, Figure 5-12). On the other hand Ctr9 and Paf1 reduce

levels of TEN1 mRNA (affecting a member of the telomere capping complex, CST)

(Pathway c, Figure 5-12) while other components of the PAF1 complex had comparatively small effects. Thus, in summary, PAF1 complex components show

complex effects on levels of three transcripts that affect telomere biology: TLC1,

VPS36 (perhaps also through TLC1 RNA regulation) and TEN1.

CDC73 is adjacent to VPS36 and the pair have been suggested to exhibit a

neighbouring gene effect. In this Chapter I confirmed that the CDC73 disruption

partially reduces VPS36 function (and by this criterion is causing a neighbouring

gene effect) (Pathway d, Figure 5-12). This is in agreement with a model where the

In addition to the neighbouring gene effect, there are other more important (trans)

interactions between the PAF1 complex and VPS36. First, Cdc73 and the other

members of the PAF1 complex are required for normal, high VPS36 mRNA levels. In

addition vps36Δ exacerbates the temperature sensitive phenotype of cdc73Δ

mutants. Furthermore, there is a synthetic lethal interaction in cells with defective

ESCRT-I, ESCRT-II and ESCRT-III machineries and ctr9Δ or paf1Δ mutations. This

suggests that the ESCRT machinery functions redundantly with Ctr9/Paf1 to maintain yeast cell viability (Pathway e, Figure 5-12).

How the PAF1 complex and the ESCRT components function together to maintain yeast cell fitness is not clear. The higher sensitivity of ESCRT deletion mutants to 6- azauracil suggested that the ESCRT pathway plays a role in transcription elongation (Song et al. 2014). Importantly, ESCRT proteins physically associate with 3’ regions of actively transcribed genes (Song et al. 2014). Thus, since the PAF1 complex

facilitates transcription, paf1Δvps36Δ and ctr9Δvps36Δ might have exacerbated

transcriptional defects that are not compatible with life.

Another hypothesis is that double mutants (carrying deletions of both PAF1 and ESCRT complex components) could have increased levels of transcription-replication fork collisions. Paf1 (together with Ino80) was shown to be important for RNA pol II degradation to avoid transcription-replication fork collision (Poli et al. 2016).

Additionally, many have shown that Rpb1 is ubiquitylated upon DNA damage, marking RNA pol II for degradation (Huibregtse et al. 1997; Beaudenon et al. 1999; Somesh et al. 2005; Chen et al. 2007). Since the ESCRT machinery is involved in the sorting of ubiquitylated proteins for degradation, it is possible that the ESCRT complex is involved in the degradation of RNA pol II. If true, the combined effects of loss of PAF1 (less signalling for RNA pol II degradation) and loss of ESCRT (less capacity to degrade RNA pol II) could cause irreversible stalling in replication forks, leading to cell cycle arrest.

Irrespective of the precise mechanism it is plausible that the synthetic lethal

interactions between paf1Δ or ctr9Δ and ESCRTcomponent deletions are based, at

least in part, on their functions at telomeres (Pathways e,f, Figure 5-12). Vps36 and the ESCRT II complex are connected with telomere biology. In this Chapter I saw

slightly higher levels of TLC1 RNA in vps36Δ strains and this correlates with telomere

genetic background, temperature, or other environmental conditions that might be different in my experiments when compared to the previously reported experiments. Indeed, telomere length can be affected by environmental conditions such as

temperature and ethanol concentration (Romano et al. 2013). The ESCRT-II complex role on telomere function might be due to a role in telomerase turnover, or in the turnover of other proteins involved in telomerase turnover/transcription repression. An important new insight comes from the observation that Paf1 and Ctr9, the critical

members of the PAF1 complex, inhibit TEN1 mRNA accumulation. It is known that in

plant and yeast cells that high levels of Ten1 can inhibit telomerase recruitment (Qian

et al. 2009a; Leehy et al. 2013). Thus a ctr9Δ mutation causing a reduction in TLC1

RNA combined with an increase in TEN1 mRNA will be more harmful to telomere

function, and cell fitness, than a cdc73Δ mutation which just affects TLC1 RNA

levels. I could only observe a very modest effect on STN1 expression (also a

telomerase repressor) in cdc73Δ strains, showing that although contributing to cell

sickness, high Ten1 levels are perhaps just a small fraction of what makes paf1Δ

cdc13-1 and ctr9Δcdc13-1 cells sick. Since the PAF1 complex is needed for proper

levels of hundreds of transcripts, it might just be that one of those altered transcripts is required at normal levels in telomere defective strains.

Finally, the interactions here described between the PAF1 complex and VPS36 might

be relevant to health in higher eukaryotes. In Drosophila, the VPS36 orthologue was

reported to affect the Hedgehog (Hh) pathway (Yang et al. 2013). The Hedgehog pathway, conserved between vertebrate and invertebrates (Nussleinvolhard and Wieschaus 1980; Huangfu and Anderson 2006), is involved in embryonic and post- embryonic development and has a strong role on neural stem cell maintenance (Ahn and Joyner 2005; Balordi and Fishell 2007; Ingham et al. 2011; Briscoe and Therond 2013). Interestingly, Ctr9 has been shown to regulate dopamine transporter

trafficking (essential for proper neural function) in mammalian cells (De Gois et al. 2015), and could therefore potentially play a role like the Hedgehog pathway in neural stem cells.

Furthermore, both Hedgehog (Vps36) and Wnt (PAF1 complex) signalling pathways are constitutively active in many cancers (Taipale and Beachy 2001) and common

Figure 5-12 Cartoon for the regulation of cell viability by the PAF1 complex.

The Paf1 complex is composed of Cdc73, Paf1, Ctr9, Leo1 and Rtf1. The Paf1

complex promotes the expression in trans of both TLC1 (a) and VPS36 (b), while

Paf1 and Ctr9 repress the expression of TEN1 (c). Additionally, CDC73 and VPS36

interact in cis(d). Telomere maintenance is essential for cell viability and Ten1 has

a repressive effect over telomere lengthening while TLC1 promotes telomere

lengthening. Therefore, the PAF1 complex, mainly through Paf1 and Ctr9, maintains cell viability by promoting telomere integrity. Ctr9 and Paf1, together with Vps36,

contribute to cell viability (e) through a mechanism that I cannot yet define, although

Thus, a better understanding of the cross-talk between the Hedgehog and Wnt pathways could be helpful. The links between the PAF1 complex, Vps36 and telomere biology in yeast may help to understand the role of the human PAF1 complex in cancer.

5.4. Future work

It would be interesting to see if TLC1 overexpression rescues the fitness defects of

paf1Δcdc13-1 and ctr9Δcdc13-1 (in comparison to cdc73Δ cdc13-1). This would

show if the low TLC1 levels (caused by deletion of CDC73, PAF1 and CTR9) have a

critical role in the low fitness of paf1Δcdc13-1 and ctr9Δcdc13-1 cells. A hypothesis

is that such expression would recover cdc73Δcdc13-1 fitness defects (at very low

6. PAF1 complex components regulate TERRA and