Since many lncRNAs are Pol II-transcribed, capped and polyadenylated (see Chapter 1), all RNA-seq samples were poly(A)-enriched (see Chapter 2 for details).
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All RNA-seq samples of RNA processing mutants used in the current study are detailed in Table 2.1. All RNA-seq samples of different physiological conditions/environmental perturbations used in the current study are detailed in Table 2.2.
For all samples, cells were grown, RNA harvested and strand-specific cDNA libraries prepared by myself (see Chapter 2). This is with the exception of: meiotic time-course samples, for which cells were grown and RNA prepared by Dr Cristina Cotobal (Mata laboratory, University of Cambridge); pka1 samples under glucose starvation, for which cells were grown by Dr Charalampos Rallis (Bähler laboratory).
3.2.1 RNA processing/ degradation mutants
An overview of RNA processing/degradation pathways has been set out in Section 1.4, Chapter 1.
A panel of RNA processing mutants was chosen to cover key aspects of RNA processing and degradation (see Table 2.1). This panel includes: exonucleases in both the nucleus and cytoplasm; cofactors of these exonucleases; factors involved in deadenylation; factors that target decapping substrates; poly(A)-binding proteins; RNA interference (RNAi) factors; and factors involved in nonsense mediated decay (NMD).
The exosome contributes to the processing, quality control and turnover of a large number of cellular RNAs in both the nucleus and the cytoplasm where it degrades RNAs in a 3’-5’ direction (see Section 1.4, Chapter 1). The exosome consists of nine inactive subunits plus an active ribonuclease, Dis3, which has both 3’-5’ exonucleolytic as well as endonucleolytic activity. Thus the mutant dis3-54 was chosen for the current study to reveal the role of the core exosome on lncRNA regulation.
Rrp6 confers nuclear specificity to the exosome, and both a temperature-sensitive mutant (rrp6-ts) and a null-mutant (rrp6) were included in the current study. In addition, null-mutants for Pab2, and TRAMP complex components Cid14 and Mtr4,
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were chosen based on their known interactions with the nuclear exosome in S. pombe. A null mutant of the poly(A)-binding protein Nab2 was also included in the current study since Nab2 has been proposed to play a role in differentiating between mRNAs and lncRNAs (Tuck and Tollervey 2013), and has also been shown to impede Pab2/Rrp6-mediated decay by competing with Pab2 for binding poly(A) tails (Grenier St-Sauveur et al. 2013).
In the cytoplasm of S. pombe, three key pathways contribute to RNA degradation: the exosome which degrades in a 3’-5’ manner and is connected to its cytoplasmic substrates via the SKI complex; the 5’-3’ exonuclease Exo2; and the recently identified 3’-5’ exonuclease Dis3L2 (Malecki et al. 2013). Mutants of all of these factors were therefore included in the current study.
The RNAi pathway in S. pombe is emerging as having roles in gene expression beyond heterochromatin formation at centromeres and telomeres, with several recent studies revealing roles for RNAi in transcriptional and post-transcriptional regulation of euchromatic loci (Cawley et al. 2004, Smialowska et al. 2014, Woolcock et al. 2011, Woolcock et al. 2012, Yamanaka et al. 2013). Moreover, connections between the RNA processing activities of the exosome and the RNAi pathway are emerging as playing a role in the regulation of lncRNAs (Gullerova and Proudfoot 2008, Lee et al. 2013, Shah et al. 2014, Zofall et al. 2009). Thus, mutants of the core RNAi components Dicer (dcr1), Argonaute (ago1) and RNA-dependent RNA polymerase (rdp1) were included in the current study.
Several double mutants for these key pathways were included to elucidate interactions between pathways in regulating lncRNA expression (see Table 2.1). The rrp6/dcr1 and exo2/dcr1 mutants will be discussed in more detail in Chapter 5.
3.2.2 Environmental perturbations/physiological conditions
Different physiologically relevant growth conditions (referred to as ‘growth conditions’) were chosen to reflect key physiologically relevant states for S. pombe.
In fission yeast, meiosis is accompanied by a complex gene expression programme in which more than 50% of the genome is regulated. Moreover, microarray studies
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have shown that changes in gene expression occur in successive expression waves throughout the course of meiosis (Mata et al. 2007, Mata et al. 2002), and RNA-seq studies have revealed the expression of several meiosis-specific lncRNAs (Bitton et al. 2011, Rhind et al. 2011, Wilhelm et al. 2008). Cells harvested at time-points throughout the course of meiosis were therefore included in the current study.
Fission yeast is emerging as a complementary model system to budding yeast for studying the molecular mechanisms for cellular ageing (Rallis et al. 2013). S. pombe undergoes a progressive decline in viability after entering a quiescent stationary phase, a phenomenon known as chronological ageing, and which is often modeled by survival in glucose starvation (Roux et al. 2006). A nutrient-signaling pathway, which includes the serine/threonine cAMP-activated protein kinase Pka1, is known to regulate this process. Pka1 is a long-lived mutant that accumulates fewer reactive oxygen species and has delayed initiation of apoptosis compared with WT cells (Roux et al. 2006).
Thus stationary WT cells at 100% and 50% survival in glucose starvation were included in the current study to determine lncRNA expression during chronological ageing. pka1 cells at 100% and 50% survival were also included to determine lncRNA expression during chronological ageing in a long-lived mutant. When WT cells were at 50% survival, pka1 cells were at 87% survival. This pka1 time-point was included in the current study and is of interest because it may help determine whether changes in lncRNA expression during chronological ageing are a function of time in stationary phase, or whether they are related to the underlying molecular mechanisms of chronological ageing.
The cellular response of fission yeast to nitrogen starvation has been extensively studied. Following the withdrawal of the nitrogen source (NH4Cl) from the culture
medium, S. pombe cells exit the cell cycle, inhibit cellular growth and enter the G0 phase, thus providing an excellent model to study cellular quiescence (Yanagida 2009). The adaptation to nitrogen starvation occurs in several stages. In the initial stage (within the first ~8h), the cell size is reduced by two subsequent cell divisions, consequently forming small round cells (Shimanuki et al. 2007). Following these two rounds of division, the cell cycle is arrested in an ‘uncommitted’ G1 phase.
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During this period, cells may undergo meiosis and produce spores, providing that a partner of the opposite mating type is available. In the absence of a mating partner, the cells lose the ability to mate after 12h and commit to the G0 phase (Yanagida et al. 2011). Within 24h of nitrogen starvation, the cellular volume, mRNA and protein content are reduced to about 55%, 20%, and 50% of the vegetative cell content, respectively (Marguerat et al. 2012). Fully adapted G0 cells can survive for months in the nitrogen starvation culture medium, exhibiting increased stress resistance. Thus nitrogen starved cells at 24h and 7 days were included in the current study to reflect distinct stages of adaptation to nitrogen starvation.