Strand-specific RNA sequencing of S. pombe reveals a highly structured programme of ncRNA expression at over 600 loci. Functional investigations show that this extensive ncRNA landscape controls the complex programme of sexual differentiation in S. pombe.
The model eukaryote S. pombe features substantial numbers of ncRNAs many of which are antisense regulatory transcripts (ARTs), ncRNAs expressed on the opposing strand to coding sequences.Individual ARTs are generated during the mitotic cycle, or at discrete stages of sexual differentiation to downregulate the levels of proteins that drive and coordinate sexual differentiation.Antisense transcription occurring from events such as bidirectional transcription is not simply artefactual ‘chatter', it performs a critical role in regulating gene expression.
Regulation of the RNA profile is a principal control driving sexual differentiation in the fission yeast Schizosaccharomyces pombe. Before transcription, RNAi-mediated formation of heterochromatin is used to suppress expression, while post-transcription, regulation is achieved via the active stabilisation or destruction of transcripts, and through at least two distinct types of splicing control (Mata et al, 2002; Shimoseki and Shimoda, 2001; Averbeck et al, 2005; Mata and Bähler, 2006; Xue-Franzen et al, 2006; Moldon et al, 2008; Djupedal et al, 2009; Amorim et al, 2010; Grewal, 2010; Cremona et al, 2011).
Around 94% of the S. pombe genome is transcribed (Wilhelm et al, 2008). While many of these transcripts encode proteins (Wood et al, 2002; Bitton et al, 2011), the majority have no known function. We used a strand-specific protocol to sequence total RNA extracts taken from vegetatively growing cells, and at different points during a time course of sexual differentiation. The resulting data redefined existing gene coordinates and identified additional transcribed loci. The frequency of reads at each of these was used to monitor transcript abundance.
Transcript levels at 6599 loci changed in at least one sample (G-statistic; False Discovery Rate <5%). 4231 (72.3%), of which 4011 map to protein-coding genes, while 809 loci were antisense to a known gene. Comparisons between haploid and diploid strains identified changes in transcript levels at over 1000 loci.
At 354 loci, greater antisense abundance was observed relative to sense, in at least one sample (putative antisense regulatory transcripts—ARTs). Since antisense mechanisms are known to modulate sense transcript expression through a variety of inhibitory mechanisms (Faghihi and Wahlestedt, 2009), we postulated that the waves of antisense expression activated at different stages during meiosis might be regulating protein expression.
To ask whether transcription factors that drive sense-transcript levels influenced ART production, we performed RNA-seq of a pat1.114 diploid meiosis in the absence of the transcription factors Atf21 and Atf31 (responsible for late meiotic transcription; Mata et al, 2002). Transcript levels at 185 ncRNA loci showed significant changes in the knockout backgrounds. Although meiotic progression is largely unaffected by removal of Atf21 and Atf31, viability of the resulting spores was significantly diminished, indicating that Atf21- and Atf31-mediated events are critical to efficient sexual differentiation.
If changes to relative antisense/sense transcript levels during a particular phase of sexual differentiation were to regulate protein expression, then the continued presence of the antisense at points in the differentiation programme where it would normally be absent should abolish protein function during this phase. We tested this hypothesis at four loci representing the three means of antisense production: convergent gene expression, improper termination and nascent transcription from an independent locus. Induction of the natural antisense transcripts that opposed spo4+, spo6+ and dis1+ (Figures 3 and 7) in trans from a heterologous locus phenocopied a loss of function of the target protein. ART overexpression decreased Dis1 protein levels. Antisense transcription opposing spk1+ originated from improper termination of the sense ups1+ transcript on the opposite strand (Figure 3B, left locus). Expression of either the natural full-length ups1+ transcript or a truncated version, restricted to the portion of ups1+ overlapping spk1+ (Figure 3, orange transcripts) in trans from a heterologous locus phenocopied the spk1.Δ differentiation deficiency. Convergent transcription from a neighbouring gene on the opposing strand is, therefore, an effective mechanism to generate RNAi-mediated (below) silencing in fission yeast. Further analysis of the data revealed, for many loci, substantial changes in UTR length over the course of meiosis, suggesting that UTR dynamics may have an active role in regulating gene expression by controlling the transcriptional overlap between convergent adjacent gene pairs.
The RNAi machinery (Grewal, 2010) was required for antisense suppression at each of the dis1, spk1, spo4 and spo6 loci, as antisense to each locus had no impact in ago1.Δ, dcr1.Δ and rdp1.Δ backgrounds. We conclude that RNAi control has a key role in maintaining the fidelity of sexual differentiation in fission yeast. The histone H3 methyl transferase Clr4 was required for antisense control from a heterologous locus.
Thus, a significant portion of the impact of ncRNA upon sexual differentiation arises from antisense gene silencing. Importantly, in contrast to the extensively characterised ability of the RNAi machinery to operate in cis at a target locus in S. pombe (Grewal, 2010), each case of gene silencing generated here could be achieved in trans by expression of the antisense transcript from a single heterologous locus elsewhere in the genome.
Integration of an antibiotic marker gene immediately downstream of the dis1+ locus instigated antisense control in an orientation-dependent manner. PCR-based gene tagging approaches are widely used to fuse the coding sequences of epitope or protein tags to a gene of interest. Not only do these tagging approaches disrupt normal 3′UTR controls, but the insertion of a heterologous marker gene immediately downstream of an ORF can clearly have a significant impact upon transcriptional control of the resulting fusion protein. Thus, PCR tagging approaches can no longer be viewed as benign manipulations of a locus that only result in the production of a tagged protein product.
Repression of Dis1 function by gene deletion or antisense control revealed a key role this conserved microtubule regulator in driving the horsetail nuclear migrations that promote recombination during meiotic prophase.
Non-coding transcripts have often been viewed as simple ‘chatter', maintained solely because evolutionary pressures have not been strong enough to force their elimination from the system. Our data show that phenomena such as improper termination and bidirectional transcription are not simply interesting artifacts arising from the complexities of transcription or genome history, but have a critical role in regulating gene expression in the current genome. Given the widespread use of RNAi, it is reasonable to anticipate that future analyses will establish ARTs to have equal importance in other organisms, including vertebrates.
These data highlight the need to modify our concept of a gene from that of a spatially distinct locus. This view is becoming increasingly untenable. Not only are the 5′ and 3′ ends of many genes indistinct, but that this lack of a hard and fast boundary is actively used by cells to control the transcription of adjacent and overlapping loci, and thus to regulate critical events in the life of a cell.
Strand-specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3′ termination and bidirectional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent ‘horsetail' stage of meiosis. Antisense production had no impact at any of these loci when the RNA interference (RNAi) machinery was removed. Thus, far from being simply ‘genome chatter', this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe.