BACKGROUND
genome-wide transcriptional studies have revealed that eukaryotic genomes are pervasively transcribed, producing thousands long  non-coding rnas   <cit> .
nevertheless, functional characterisation of lncrnas is lagging considerably behind their discovery rate  <cit> .
one reason for this discrepancy is the fact that only a small proportion of these molecules are essential for life as shown by knockout studies of mammalian lncrnas  <cit> .
therefore, the cellular effects of a mutated lncrna might be masked by other factors with compensatory functions, indicating that the role of lncrnas should be interrogated under synthetic knockout conditions.
another challenge for determining lncrna functions stems from the fact that genetic manipulations of certain lncrnas often affect the expression of other overlapping genes  <cit> .
this issue is, of course, less true for long intergenic non-coding rnas , which do not overlap any other genomic features.
in saccharomyces cerevisiae, approximately 85% of the genome is transcribed, generating a large number of lncrnas  <cit>  that are reminiscent of those found in higher eukaryotes  <cit> .
among these non-coding transcripts there are classes of lncrnas sensitive to rna decay machinery, such as cryptic unstable transcripts, xrn1-sensitive unstable transcripts and telomeric repeat-containing rnas , as well as lncrnas expressed only under specific conditions such as meiotic unannotated transcripts  <cit> , cytoplasmically degraded-cryptic unstable transcripts  <cit> , stress-inducible lncrnas  <cit>  and telomerase mutant lncrnas  <cit> .
in addition, there are stable lncrnas in wild-type yeast appropriately defined as stable unannotated transcripts , which represent approximately 12% of the yeast transcriptome  <cit> .
suts evade degradation in the nucleus and are processed in the cytoplasm in a similar manner to mrnas  <cit> ; thus, it was suggested that these transcripts might be functionally important  <cit> .
only a handful of suts have been experimentally investigated and most of these have been assigned roles in regulating gene expression.
in fact, it is the transcriptional process of some of these characterised lncrnas that has a functional output and not the transcripts themselves  <cit> .
for example, transcription of the lncrna designated as irt <dig>  represses the expression of its adjacent ime <dig> protein-coding gene by establishing repressive chromatin modifications at the ime <dig> promoter  <cit> .
additionally, transcription of the gal <dig> lncrna  recruits similar chromatin modifying activities to alter nucleosome occupancy and to repress the expression of gal <dig> and gal <dig>  <cit> .
likewise, the transcription of two other lncrnas, icr <dig> and pwr <dig>  modulates chromatin structure and transcription factor binding at the flo <dig> promoter  <cit> .
other steady-state yeast lncrnas control gene expression through transcriptional interference and not through recruitment and modulation of chromatin modifications.
for instance, transcription of the ime4-antisense lncrna blocks sense strand transcription of the ime <dig> gene  <cit>  and transcription of the srg <dig> lncrna prevents transcription initiation at the ser <dig> promoter  <cit> .
a common feature of almost all characterised yeast lncrnas, including the suts described above, is that they function in cis by regulating their cognate genes.
therefore, it remains unclear whether a significant number of yeast lncrnas could also function in trans, distant from the locus from which they are transcribed, in order to regulate gene expression and other dna-based processes.
the rna component of the telomerase complex, known as telomerase component  <dig>   <cit> , constitutes the single most characterised yeast lincrna that functions in trans.
tlc <dig> has a functional orthologue in human cells, known as terc  <cit> , but these two rnas vary in size and nucleotide sequence.
tlc <dig> is transcribed on chromosome ii and then interacts physically with proteins est <dig>  est <dig> and est <dig> to form the telomerase complex, whose role is to solve the end replication problem by synthesising telomeric dna repeats and preventing telomere shortening during each cell division  <cit> .
tlc <dig> is recruited to telomeres through a chain of physical interactions coordinated by the heterodimeric ku complex  .
once at the telomeres, tlc <dig> functions as the template for telomere dna synthesis by est <dig>  the reverse transcriptase component of the telomerase complex  <cit> .
in the absence of functional telomerase, telomeres are significantly shortened and cells stop dividing and senesce  <cit> .
within a population of senescing cells some survivors arise by fixing their telomere length through a dna recombination-based mechanism  <cit> .
this alternative telomere maintenance mechanism is mediated by two distinct rad52-dependent dna recombination pathways; a commonly induced type i pathway, which requires the rad <dig>  rad <dig>  rad <dig> and rad <dig> proteins, or a type ii pathway, which involves the trimeric mrx complex  and rad <dig>  <cit> .
additional proteins, including the ino <dig> chromatin remodelling complex, function as regulators of the telomere recombination mechanisms  <cit>  in order to preserve telomere length and function.
telomere homeostasis is also dependent on the presence of 3’-end single stranded dna  at chromosome ends, known as telomeric overhangs.
in most organisms, the telomeric overhangs consist of g-rich repeats and extend over the c-rich strands at both ends of a chromosome  <cit> .
telomeric overhangs are necessary during telomere replication because they provide a substrate for the rna moiety of the telomerase complex.
formation of telomeric overhangs is closely linked to dna replication, occurs independently of telomerase action and is regulated by evolutionary conserved mechanisms  <cit> .
in s. cerevisiae, various exonucleolytic activities are involved in the formation of telomeric ssdna, including the mrx complex and the 5’–3’ double-strand-specific exonuclease exo <dig>  <cit> .
the nuclease activity of these enzymes towards chromosome ends is inhibited by telomeric capping factors, such as rap <dig>  rif <dig> and rif <dig>  that block mrx access, and the cst  and ku complexes, which protect telomeres from exo1-mediated degradation  <cit> .
defects in telomere capping that can impact on the activity of exonucleases towards telomere ends lead to various problems, including premature senescence, cell-cycle arrest and accumulation of telomeric ssdna .
hence, complete understanding of the factors and mechanisms that control telomere-end processing is important for determining how telomeres maintain their structure and function  <cit> .
in this study, we sought to identify genetic interactions  between yeast lincrnas and protein-coding genes in order to evaluate their functional relationships using synthetic genetic array  technology.
as proof of principle, we initially demonstrated that the gi network of the lincrna tlc <dig> is consistent with its function in telomere maintenance.
therefore, we next applied the same systematic genetic approach to six other uncharacterised intergenic suts.
interestingly, the gi profile of sut <dig> connected this lincrna to telomere organisation and follow-up experiments established sut <dig> as a novel factor of telomere overhang homeostasis.
this study proposes that systematic analysis of gis could unveil the function of lincrnas in s. cerevisiae and other complex organisms.
RESULTS
the gi network of tlc <dig> corresponds to its cellular function
although genome-wide transcriptional studies have revealed an enormous amount of non-coding rnas that can be synthesised from the s. cerevisiae genome, a relatively small number of these lncrnas have been functionally characterised  <cit> .
we reasoned that we could obtain insights about the function of yeast lincrnas by comprehensively mapping their gis through an approach that has been previously applied for protein-coding genes  <cit> .
therefore, we employed the sga methodology to construct double mutants in which the deletion of an intergenic sut  is systematically combined with individual deletions of non-essential genes in budding yeast.
gis are scored in double mutants that show significant deviation in fitness compared to the growth of the corresponding single gene deletion strains generated during the control sga screen .
specifically, a negative gi  refers to a more severe fitness defect in the double mutant compared to the corresponding single gene deletion mutants, while a positive gi  corresponds to growth with a less severe fitness defect in the double mutant in comparison to the single deletion mutants.
the precise stepwise procedure used for the sga screens and the deviations from a conventional approach  <cit>  are shown in fig.
1a .fig.
<dig>
tlc <dig> validates the sga approach for interrogating the function of lincrnas.
a schematic illustration of the sga screening procedure used for studying lincrnas.
a query strain  carrying deletion of a lincrna  is mated to an array of  <dig> single knockout strains , each lacking a non-essential protein-coding gene .
following several selection steps, double mutant strains are isolated and their growth fitness is compared to the corresponding single deletion strains derived from a parallel control sga screen.
b genetic interactions  of tlc <dig> associated with the three indicated gene ontology  biological processes.
gene nodes are coloured according to their assigned go annotation.
negative  and positive  gis that belong to the three go terms are shown.
previously established tlc <dig> cofactors are highlighted in light grey boxes.
c venn diagram showing the significant overlap  between genes classified as having a negative gi with tlc <dig> and est <dig> in this study.
the p value was generated using the hypergeometric test
to demonstrate proof of concept for the aforementioned rationale, we initially applied the sga procedure to the yeast lincrna tlc <dig>  which has a well-defined cellular role in telomere maintenance  <cit> .
the sga screen was carried out in duplicate by combining the tlc1Δ strain against an ordered array of approximately  <dig> viable protein-coding gene deletion strains.
a total of  <dig> ngis and  <dig> pgis of tlc <dig> were identified within the two sga screens .
we identified ngis with ku complex components  whose synthetic lethal interactions with tlc <dig> were previously reported  <cit> .
moreover, tlc <dig> showed ngis with genes  whose proteins are integral components of the rad52-mediated telomere recombination pathway  <cit>  and the ino <dig> chromatin remodelling complex that is also implicated in telomere lengthening via homologous recombination  <cit>  .
since pgis are typically detected among components of the same protein complex  <cit> , we also found that tlc <dig> has a positive gi with its co-factors est <dig> and est <dig>  which are subunits of the telomerase complex.
to further support the relevance of the identified gis with the function of tlc <dig>  we then performed gene ontology  analysis of our sga data, as an objective metric for deriving functional utility from gi datasets  <cit> .
we chose to perform go enrichment analysis using only ngis, since the sga technique demonstrates higher precision rate on detecting true ngis compared to true pgis and, in addition, ngis frequently occur between genes with overlapping functions  <cit> .
in agreement with the individual gis mentioned above, go analysis of all tlc <dig> ngis significantly enriched the biological process terms, telomere organisation  and dna recombination  .
furthermore, tlc <dig> ngis significantly enriched the term chromatin remodelling , a cellular activity that has been associated with telomere maintenance  .
these results show that the sga-derived gis of tlc <dig> are consistent with the cellular function of this yeast lincrna.fig.
<dig> genetic interactions link intergenic suts to diverse biological processes.
a heatmap of gene ontology  terms enriched by genes whose deletions result in negative genetic interactions.
the synthetic genetic array  procedure was performed for tlc <dig>  est <dig> and six intergenic suts.
the negative genetic interaction  from the different sga screens were analysed as described in methods to identify enriched go terms with p ≤  <dig>  .
b and c heatmap of go terms enriched by genes classified as having a ngi with tlc <dig> , sut <dig>  or their corresponding flanking genes.
the ngis of the genes flanking the tested lincrnas were extracted from the drygin database  and szappanos et al.
<cit>
it was previously demonstrated that genes with highly similar gi profiles have strong functional relationship to the extent that they can be physical partners within the same complex  <cit> .
hence, we hypothesised that tlc <dig> should have a gi profile that is highly correlated to the profile of other components of the telomerase complex.
to test this hypothesis and support the validity of our tlc <dig> sga screens, we performed an independent sga screen for the protein-coding gene est <dig>  which encodes a subunit of the telomerase complex.
we identified  <dig> ngis for est <dig>  of which  <dig> interactions were common with tlc <dig> ngis , indicating that their gi profiles are highly related .
the shared gis between est <dig> and tlc <dig> enrich for the go terms telomere organisation and dna recombination that are related to their cellular function .
to further examine the validity of our sga screens, we also compared our identified tlc <dig> gis to est <dig> gis reported in previous studies.
notably, about 40% of tlc <dig> ngis identified in this study were also identified as est <dig> negative gis by two independent sga studies  <cit> , and once again, the common ngis between tlc <dig> and est <dig> enrich the go terms telomere organisation and dna recombination .
overall, these findings demonstrate that sga-derived gis can illuminate the biological role of tlc <dig>  and support the utility of this approach in identifying the function of other uncharacterised yeast lincrnas.
sga analysis implicates intergenic suts in diverse cellular processes
the s. cerevisiae genome encodes approximately  <dig> suts  <cit> , of which  <dig> are intergenic  since they do not overlap any other known genomic features .
we applied the sga procedure to six intergenic suts that differ in size, distance from adjacent genes and level of expression  <cit>  .
the sga screen for each deleted lincrna was paired with a control sga screen  to minimise the ‘batch effect’  <cit> .
we identified in total  <dig> negative gis and  <dig> positive gis for all six tested suts .
we subsequently analysed the ngis of each sut  for the enrichment of go biological process terms as described for tlc <dig> above.
notably, all tested lincrnas enriched at least one go term with the exception of sut <dig> which did not highlight any terms despite displaying  <dig> ngis .
it is possible that this lincrna is involved in multiple processes that are diluted among the identified gis.
the other five suts exhibited ngis that result in distinct go enrichment profiles .
in particular, the sga screen using the sut457Δ strain enriched for the two go terms, telomere organisation  and not-yet-annotated .
the latter term consists of functionally uncharacterised genes and therefore does not provide any insights into the function of this lincrna.
furthermore, sut <dig> also showed a specific profile since its screen enriched go terms  associated with vesicle function .
on the other hand, the other three lincrnas, sut <dig>  sut <dig> and sut <dig>  exhibited go profiles that are more diverse in function.
sut <dig> and sut <dig> sga screens each enriched seven different biological processes, while sut <dig> showed the most varied profile with enrichment of ten different go terms .
the heterogeneous go term profiles suggest that the latter three suts are pleiotropic or that the enriched cellular processes are interconnected  <cit> .
altogether, these results indicate that, under physiological conditions, intergenic suts are implicated in a broad spectrum of biological processes that are important for normal cell growth.
sut <dig> and sut <dig> exhibit distinct gi profiles in comparison to their adjacent genes
previously characterised suts and other yeast non-coding rnas have been linked to transcriptional regulation of their neighbouring genes  <cit> .
therefore, we reasoned that sga analysis of lincrnas, which control their cognate genes, will lead to the discovery of gis which would be similar to those identified for their neighbouring genes.
to compare the gi networks of the examined intergenic suts with those of their neighbouring genes, we exploited the gi data of protein-coding genes available on the drygin dataset  and those generated by szappanos et al.
<cit> , which are compatible with the sga approach used in this study.
we first compared the go enrichment profiles of tlc <dig> with its upstream and downstream gene, pdx <dig> and csg <dig>  respectively, since tlc <dig> is not linked to the transcriptional control of its neighbours.
as predicted, the gis of tlc <dig> enrich go terms that are distinct to those enriched by the gis of its flanking genes .
similarly to tlc <dig>  two other lincrnas, sut <dig> and sut <dig>  exhibit go enrichment profiles that are completely different from those of their upstream and downstream neighbouring genes .
in contrast, sut <dig>  sut <dig> and sut <dig>  whose gis enrich diverse biological processes, display go profiles that partially overlap those of their adjacent genes .
these findings suggest that sut <dig>  sut <dig> and sut <dig> might have functions that are linked to the expression of their neighbouring genes, while sut <dig> and sut <dig> have roles that are independent to the function of their adjacent genes.
deletion of sut <dig> accelerates senescence in telomerase-deficient cells
sut <dig> showed a very specific go enrichment profile that was distinct from its flanking genes  and, therefore, we decided to further characterise its cellular role.
initially, we verified by northern blot analysis the loss of sut <dig> transcript  in the sut457Δ strain which was processed in the sga screens .
then, we examined whether construction of the sut457Δ strain genetically perturbs the expression of its neighbouring genes .
quantitative rt-pcr analysis showed that in sut457Δ cells the expression of sut457-adjacent genes syp <dig>  snr <dig> and rps14a remains unaffected compared to a wild-type control strain .
this result demonstrates that construction of sut <dig> deletion does not lead to the previously reported neighbouring gene effect  <cit>  and, hence, it confirms that the gi network of sut457Δ is directly associated with sut <dig> itself and not with its adjacent genes.fig.
<dig> deletion of sut <dig> accelerates senescence in telomerase-deficient cells.
a schematic representation of the sut <dig> locus on chromosome iii.
arrows indicate direction of transcription.
b expression levels of syp <dig>  sut <dig>  snr <dig> and rps14a determined by qrt-pcr using total rna extracted from isogenic wild-type and sut457Δ strains.
the expression levels of each gene were normalised to the expression of act <dig>  error bars represent standard error of the mean, resulting from three independent replicates.
*** p <  <dig> ; n/s, not significant .
c genetic interactions of sut <dig> associated with the gene ontology term telomere organisation.
gene names in bold represent negative genetic interactions while gene names in regular font correspond to positive genetic interactions.
d telomere length analysis of genomic dna isolated from the indicated wild-type and mutant strains at passage  <dig>  the extracted dna was fragmented with xhoi and subjected to denatured southern blotting using a biotinylated probe against telomeric repeats.
e senescence assays performed using liquid cultures of the indicated isogenic wild-type and mutant strains.
the strains were generated through tetrad dissection of the heterozygous diploid double mutant sut457/sut457Δtlc1/tlc1Δ.
this plot represents one of three independent tetrads examined .
f telomere length analysis performed as in  using genomic dna isolated from the indicated wild-type and mutant strains during passages  <dig> and 8
sut <dig> genetically interacts with  <dig> out of  <dig> telomere organisation genes  found in the sga deletion collection .
these genes are associated with roles in supporting telomerase function  , modulating subtelomeric chromatin changes   <cit> , regulating telomere-end processing  , and controlling telomere maintenance via recombination   <cit> .
the genetic link between sut <dig> and telomere organisation initially prompted us to determine if loss of this lincrna leads to changes in telomere length.
to do this, we used denaturing southern analysis and found that deletion of sut <dig> alone does not affect telomere length compared to an isogenic wild-type strain .
then, we examined whether sut457Δ, in combination with deletion of any of its  <dig> genetic interactors , affects telomere length.
as expected, significantly shorter telomeres were detected in the yku70Δ and mre11Δ single mutants compared to wild-type cells, but telomere length was not further affected in the yku70Δsut457Δ or mre11Δsut457Δ double mutants .
consistently, sut457Δ, in combination with deletion of the remaining telomere-related genetic interactors , does not have a significant change in telomere length compared to its isogenic single deletion mutants .
based on the fact that some of the telomere-associated gis of sut <dig>  are known to affect entry into senescence in telomerase-deficient cells  <cit> , we next sought to determine if deletion of sut <dig> influences the rate of senescence in tlc <dig> null cells.
therefore, we performed liquid senescence assays using the four isogenic strains, namely wild-type, sut457Δ, tlc1Δ and the double mutant sut457Δtlc1Δ.
consistent with the fact that single deletion of sut <dig> had wild-type telomeric length , we observed that sut457Δ and wild-type strains had a very similar growth profile in senescence assays .
however, we found that the double mutant sut457Δtlc1Δ had an accelerated entry into senescence compared to the tlc1Δ single mutant .
interestingly, this faster induction of senescence correlates with the appearance of shorter telomeres in the sut457Δtlc1Δ double deletion strain compared to the tlc1Δ single mutant .
moreover, deletion of sut <dig> did not affect the generation of type ii survivors in strains lacking telomerase .
altogether, these findings link the function of sut <dig> to telomere organisation and specifically show that sut <dig> participates within a telomerase-independent pathway.
loss of sut <dig> leads to accumulation of telomeric single-stranded dna
it has been previously reported that faster senescence in telomerase-negative cells can be attributed to abnormal accumulation of telomeric ssdna  <cit> .
thus, we hypothesised that deletion of sut <dig> may lead to the accumulation of ssdna at telomeres.
this hypothesis was further supported by the sga findings above, which show that sut <dig> genetically interacts with the genes yku <dig>  mre <dig>  tgs <dig>  cgi <dig>  hsc <dig> and gbp <dig>  that encode for factors implicated in telomere-end protection and in the formation of telomeric ssdna overhang  <cit> .
to examine this hypothesis, we extracted genomic dna from isogenic wild-type and sut457Δ cells at an early passage and performed native southern blotting using a c-rich oligonucleotide probe.
in addition, we extracted genomic dna from a later passage to mimic the subculturing of cells resulting from the serial pinning steps performed during the sga procedure.
absence of sut <dig> led to an increase in the signal of telomeric ssdna compared to wild-type cells in passage  <dig> , which became even more intense after subculturing yeast cells for five passages  .
in order to verify that the detected ssdna signal is due to an increase in 3’ terminal overhang and not due to accumulation of internal dna replication intermediates, we treated the native dna with bacterial exonuclease i that can only degrade terminal ssdna in a 3’ to 5’ direction.
the single stranded dna that accumulated in sut457Δ mutant was sensitive to bacterial exonuclease i digestion , indicating that the detected ssdna indeed corresponds to telomeric 3’ overhang.
we also validated the increase in telomeric overhang by comparing the levels of ssdna among  <dig> different sut457Δ mutant clones and their corresponding wild-type strains.
as above, we observed statistically significant accumulation of telomeric ssdna in sut457Δ strains at an early passage  which became even more substantial at a later passage  .fig.
<dig> loss of sut <dig> leads to exo1-dependent accumulation of telomeric ssdna.
a analysis of telomeric ssdna overhangs in isogenic wild-type and sut457Δ strains.
yeast colonies were restreaked on agar plates and genomic dna was extracted at passages  <dig>  and  <dig> , fragmented with xhoi and subjected to native southern blotting  using a biotinylated probe against telomeric repeats.
the southern blot was then treated with  <dig>  n naoh and the denatured dna was re-probed to monitor equal loading .
b accumulation of telomeric ssdna in eleven sut457Δ strains compared to their respective wild-type strains during passages  <dig> and  <dig> .
the ssdna signal for each wild-type and mutant strain detected in the native southern blot was normalised to the corresponding signal in the denatured southern blot.
error bars represent standard error of the mean.
* p <  <dig> ; ** p <  <dig> ; n/s, not significant .
c cell growth was monitored for the indicated isogenic strains at passages  <dig> and  <dig>  d analysis of telomeric ssdna levels at passage  <dig> of wild-type, sut457Δ, exo1Δ and exo1Δsut457Δ isogenic strains generated through dissection of the heterozygous diploid double mutant exo1/exo1Δsut457/sut457Δ.
southern blotting was performed as in  above.
e telomeric ssdna levels analysed at passage  <dig> in the indicated isogenic wild-type and mutant strains generated from three independent tetrads .
the quantification of the ssdna signal for each wild-type and mutant strain was performed as in  above.
error bars represent standard error of the mean.
** p <  <dig> ; n/s, not significant.
the statistical analysis was performed using one-way anova
accumulation of telomeric ssdna has been proposed to be a signal for cell cycle arrest  <cit> .
however, we did not observe a growth reduction in the sut457Δ mutant strain compared to wild-type cells .
it was previously demonstrated that growth arrest associated with telomeric overhang accumulation is masked by telomeric ssdna binding proteins like gbp <dig>  <cit> .
interestingly, gbp <dig> was identified as a sut <dig> ngi  and, therefore, we next examined how lack of gbp <dig> can affect the growth of sut457Δ cells.
we observed a strong growth arrest of gbp2Δsut457Δ cells at passage  <dig> , which coincides with the robust accumulation of telomeric ssdna .
altogether, these data suggest that sut <dig> controls the levels of telomeric ssdna overhang in order to maintain proper cellular growth.
exo <dig> nuclease is required for the accumulation of telomeric ssdna in sut457Δ cells
the 5’–3’ exonuclease exo <dig> plays a key role in telomere g-rich overhang formation in yeast by mediating c-rich strand degradation  <cit> .
the action of exo <dig> is blocked by the ku complex since lack of yku <dig> leads to unprotected telomere ends and results in exo1-dependent accumulation of telomeric ssdna  <cit> .
because sut <dig> genetically interacts with yku <dig> , we hypothesised that exo <dig> activity may be responsible for the accumulation of telomeric overhang in sut457Δ cells.
to test this hypothesis, we wanted to generate an exo1Δsut457Δ double mutant strain by crossing the exo1Δ and sut457Δ single mutants, but we realised that exo <dig> was not properly deleted in the exo1Δ strain found in the sga knockout library.
therefore, we constructed a new exo1Δ deletion strain, mated it to the sut457Δ mutant and generated isogenic haploid strains that were analysed for telomeric ssdna levels.
consistent with the above findings, we detected a strong signal in sut457Δ corresponding to the accumulation of telomeric ssdna after subculturing yeast for  <dig> passages .
notably, the telomeric overhang signal in the exo1Δsut457Δ double mutant cells is reduced back to wild-type levels .
this result was verified by quantifying the levels of telomeric ssdna in isogenic mutant strains generated from three independent tetrads .
furthermore, deletion of exo <dig> in gbp2Δsut457Δ cells rescued their growth arrest at passage  <dig> suggesting that exo1Δ suppresses the effect of sut457Δ .
altogether, these findings show that sut <dig> functions within an exo1-dependent pathway to affect telomeric overhang accumulation.
the lincrna sut <dig> acts in trans to regulate the levels of telomeric ssdna
the gi profile of sut <dig> was distinct from that of its adjacent genes  and this is also consistent with the fact that loss of sut <dig> does not affect the expression of its neighbours .
therefore, we hypothesised that, unlike most characterised yeast lncrnas, which control the expression of their cognate genes, sut <dig> may function in trans distant from its locus of synthesis.
to address this hypothesis, we examined whether ectopic expression of sut <dig> could rescue the phenotype of telomeric overhang accumulation observed in sut457Δ cells .
for this purpose, we deleted sut <dig> from its endogenous locus on chromosome iii and re-introduced it on chromosome v under the control of the ura <dig> promoter .
we also constructed another strain in which sut <dig> was ectopically expressed at the ura <dig> locus in wild-type cells to serve as an additional control .
native southern blot analysis indicated that deletion of sut457Δ from its endogenous locus results in robust increase in telomeric ssdna at passage  <dig> as previously shown , but concomitant ectopic expression of sut <dig> reduces the telomeric overhang signal to corresponding wild-type levels .
this result was verified by quantifying the levels of telomeric ssdna in five independent constructed strains .
collectively, these findings demonstrate that sut <dig> can control the levels of telomeric ssdna overhang regardless of its genomic location, indicating that this lincrna functions in trans.fig.
<dig> sut <dig> regulates the levels of telomeric ssdna by acting in trans.
a schematic illustration indicating the presence of endogenous  and ectopic sut <dig>  in wild-type and sut457Δ strains.
the level of sut <dig> expression for each constructed strain is indicated on the right: + , +++ , and – .
arrows indicate the direction of sut <dig> transcription at each locus.
b analysis of telomeric ssdna levels in wild-type , wild-type  , sut457Δ , and sut457Δ   strains at passage  <dig>  genomic dna was extracted, fragmented with xhoi and subjected to native southern blotting using a biotinylated probe against telomeric repeats .
the southern blot was then treated with  <dig>  n naoh and the denatured dna was re-probed to monitor equal loading .
c telomeric ssdna levels were analysed in five independent clones of the indicated wild-type and mutant strains at passage  <dig>  the ssdna signal for each strain detected in the native southern blot was normalised to the corresponding signal in the denatured southern blot.
error bars represent standard error of the mean.
* p <  <dig> ; n/s, not significant.
statistical analysis was performed using one-way anova
discussion
despite the plethora of long non-coding rnas identified by genome-wide approaches, rigorous genetic studies that could aid in the functional characterisation of individual lncrnas are missing  <cit> .
here, we took advantage of the genetic tractability of yeast and employed a high-throughput sga methodology to systematically catalogue the gis of six intergenic suts.
initially, we verified the utility of the approach by demonstrating that gis identified for the telomerase rna tlc <dig> enrich go terms corresponding to its known cellular function.
subsequent sga screens and analysis of gi profiles implicated the six tested intergenic suts in diverse biological processes.
one of these lincrnas, sut <dig>  exhibited gis that enriched a definitive go annotation linking it to telomere biology.
further functional characterisation unveiled the role of sut <dig> in preventing the accumulation of telomeric ssdna.
moreover, phenotypic rescue experiments showed that sut <dig> acts in trans to maintain physiological levels of telomeric ssdna.
our work reveals that systematic mapping of the gis of lncrnas could associate these molecules with specific biological processes and pinpoint their individual functions.
in yeast, the function of identified lncrnas has been mainly linked to the regulation of gene expression.
most lncrnas characterised so far regulate their cognate genes either through transcriptional interference  <cit>  or by modulating their local chromatin structure .
however, emerging evidence suggest that lncrnas could have roles beyond gene regulation  <cit> .
the go enrichment profiles of the tested intergenic suts have linked them to nuclear functions such as dna repair, meiosis and telomere organisation but also to cytoplasmic processes including membrane fusions and vesicle formation, for example, in the case of sut <dig> .
the latter observation is consistent with the fact that a large number of suts are transported to the cytoplasm and have been proposed to represent functional transcripts within this cell compartment  <cit> .
moreover, human lncrnas are co-expressed with cytoplasmic membrane proteins  <cit>  and are present within extracellular vesicles  <cit> .
therefore, future in-depth characterisation of lincrnas, like sut <dig>  could elucidate the role of these molecules in cytoplasmic processes such as vesicle biogenesis and trafficking.
only rare examples of trans-acting yeast lncrnas have been reported to date.
specifically, the pho <dig> antisense lncrnas and a cryptic unstable transcript associated with ty <dig> retrotransposons control transcriptional silencing of their target genes in trans  <cit> .
we show here that gi profiling could also provide insights about the mode of action of lincrnas in addition to inferring their biological function.
this is achieved by comparing the gi profiles of lincrnas with those of their adjacent protein-coding genes.
for example, tlc <dig> has a gi network that completely differs from that of its upstream and downstream genes  which is in line with the fact that tlc <dig> has a function unrelated to its neighbours and acts in trans at chromosome ends  <cit> .
similarly, sut <dig> and sut <dig> have distinct go profiles compared to their neighbouring genes , suggesting that these suts may also work in trans.
accordingly, sut <dig> deletion does not affect the expression of its neighbouring genes  and regulates a telomeric process occurring distal from its transcriptional site .
however, there is still a possibility that the sut <dig> locus is proximal to a gene involved in telomere-end processing within the three-dimensional architecture of the genome.
hence, in such a scenario, sut <dig> would have a role in telomere-end processing by controlling its spatially proximal gene.
nevertheless, the phenotypic rescue obtained by expressing sut <dig> from a different chromosome  makes this scenario rather unlikely.
three of the other intergenic suts tested  exhibit gis with go enrichment profiles that overlap those of their flanking genes , indicating that these lincrnas may be involved in the regulation of their nearby genes in cis.
of course, we cannot eliminate, at this point, the likelihood that construction of these three sut deletions leads to a neighbouring gene effect  <cit> , which might be responsible for the observed overlap among enriched go profiles .
we note that two of the six tested intergenic suts  exhibit gi profiles consistent with a trans-acting role and, therefore, in contrast to current evidence in the literature, we anticipate that a considerable number of budding yeast lincrnas would function at sites distal from their locus of synthesis.
lack of sut <dig> exhibited accelerated senescence in telomerase-negative cells  associated with enhanced telomere shortening  and an accumulation in telomeric ssdna overhang .
interestingly, analogous phenotypes have been reported for mutations of factors involved in telomere-end protection  <cit> .
consistent with the above observations, sut <dig> shows a positive gi with the telomere-capping factor yku <dig> , implying that these two molecules affect the same molecular process  <cit> .
notably, yku <dig> binds to and protects telomeres from exo <dig> nucleolytic processing, thus preventing the accumulation of telomeric ssdna overhang  <cit> .
our data also show that the exo <dig> nuclease is required for the increase in telomeric ssdna detected in the absence of sut <dig> .
furthermore, in agreement with the fact that exo1-dependent accumulation of telomeric ssdna induces cell-cycle arrest  <cit> , we show that loss of the telomeric ssdna masking protein gbp <dig> in sut457Δ cells results in growth inhibition after subculturing  and, importantly, this growth arrest is rescued by deletion of exo <dig> .
based on the above evidence, we speculate that sut <dig> functions in a pathway that protects telomere-ends from nucleolytic processing in order to regulate the levels of telomeric ssdna overhangs.
interestingly, the telomere-associated lncrna telomeric repeat-containing rna has also been implicated in this pathway but instead facilitates the nuclease activity of exo <dig> at chromosome ends  <cit> .
the primary nucleotide sequence of sut <dig>  does not display significant sequence similarity with any other regions within the s. cerevisiae genome and lacks conservation even amongst closely related yeast species .
nevertheless, considering its unveiled role in the fundamental and conserved cellular process of telomere overhang homeostasis  <cit> , we believe that functional analogues of sut <dig> may exist in other eukaryotes.
likewise, tlc <dig> is a lncrna whose primary sequence is not evolutionarily conserved but which has functional analogues in other organisms  <cit> .
CONCLUSIONS
transcriptome studies have enabled the discovery of a large number of lncrnas in various species.
however, it remains unclear how many of these lncrnas serve a biological function or are a mere result of transcriptional noise  <cit> .
we show here that systematic analysis of pairwise gis can provide insights about the function of individual lncrnas.
the functional maps obtained by gis could complement the information derived by other high-throughput approaches  <cit>  to help unravel the biological significance of lncrnas.
although the present study focuses on stable lincrnas, future work could also analyse gis for yeast antisense lncrnas since methodologies are being developed that abrogate the transcription of a specific non-coding rna without disrupting the expression of its associated sense mrna  <cit> .
furthermore, global mapping of gis for lncrnas can be conceivably extended to complex organisms because recent advances in rnai and crispr/cas technologies allow for efficient pairwise gene disruptions in human cells .
such analyses may unveil interspecies conservation of gi maps  <cit>  that could shed light on the evolution and functional conservation of lncrnas  <cit> .

 genome-wide transcriptional studies have revealed that eukaryotic genomes are pervasively transcribed, producing thousands long non-coding rnas. nevertheless, functional characterisation of lncrnas is lagging considerably behind their discovery rate. one reason for this discrepancy is the fact that only a small proportion of these molecules are essential for life as shown by knockout studies of mammalian lncrnas. therefore, the cellular effects of a mutated lncrna might be masked by other factors with compensatory functions, indicating that the role of lncrnas should be interrogated under synthetic knockout conditions. another challenge for determining lncrna functions stems from the fact that genetic manipulations of certain lncrnas often affect the expression of other overlapping genes. this issue is, of course, less true for long intergenic non-coding rnas, which do not overlap any other genomic features. in saccharomyces cerevisiae, approximately 85% of the genome is transcribed, generating a large number of lncrnas that are reminiscent of those found in higher eukaryotes. among these non-coding transcripts there are classes of lncrnas sensitive to rna decay machinery, such as cryptic unstable transcripts, xrn1-sensitive unstable transcripts and telomeric repeat-containing rnas, as well as lncrnas expressed only under specific conditions such as meiotic unannotated transcripts, cytoplasmically degraded-cryptic unstable transcripts, stress-inducible lncrnas and telomerase mutant lncrnas. in addition, there are stable lncrnas in wild-type yeast appropriately defined as stable unannotated transcripts, which represent approximately 12% of the yeast transcriptome. suts evade degradation in the nucleus and are processed in the cytoplasm in a similar manner to mrnas; thus, it was suggested that these transcripts might be functionally important. only a handful of suts have been experimentally investigated and most of these have been assigned roles in regulating gene expression. in fact, it is the transcriptional process of some of these characterised lncrnas that has a functional output and not the transcripts themselves. for example, transcription of the lncrna designated as irt represses the expression of its adjacent ime protein-coding gene by establishing repressive chromatin modifications at the ime promoter. additionally, transcription of the gal lncrna recruits similar chromatin modifying activities to alter nucleosome occupancy and to repress the expression of gal and gal. likewise, the transcription of two other lncrnas, icr and pwr modulates chromatin structure and transcription factor binding at the flo promoter. other steady-state yeast lncrnas control gene expression through transcriptional interference and not through recruitment and modulation of chromatin modifications. for instance, transcription of the ime4-antisense lncrna blocks sense strand transcription of the ime gene and transcription of the srg lncrna prevents transcription initiation at the ser promoter. a common feature of almost all characterised yeast lncrnas, including the suts described above, is that they function in cis by regulating their cognate genes. therefore, it remains unclear whether a significant number of yeast lncrnas could also function in trans, distant from the locus from which they are transcribed, in order to regulate gene expression and other dna-based processes. the rna component of the telomerase complex, known as telomerase component, constitutes the single most characterised yeast lincrna that functions in trans. tlc has a functional orthologue in human cells, known as terc, but these two rnas vary in size and nucleotide sequence. tlc is transcribed on chromosome ii and then interacts physically with proteins est est and est to form the telomerase complex, whose role is to solve the end replication problem by synthesising telomeric dna repeats and preventing telomere shortening during each cell division. tlc is recruited to telomeres through a chain of physical interactions coordinated by the heterodimeric ku complex. once at the telomeres, tlc functions as the template for telomere dna synthesis by est the reverse transcriptase component of the telomerase complex. in the absence of functional telomerase, telomeres are significantly shortened and cells stop dividing and senesce. within a population of senescing cells some survivors arise by fixing their telomere length through a dna recombination-based mechanism. this alternative telomere maintenance mechanism is mediated by two distinct rad52-dependent dna recombination pathways; a commonly induced type i pathway, which requires the rad rad rad and rad proteins, or a type ii pathway, which involves the trimeric mrx complex and rad. additional proteins, including the ino chromatin remodelling complex, function as regulators of the telomere recombination mechanisms in order to preserve telomere length and function. telomere homeostasis is also dependent on the presence of 3’-end single stranded dna at chromosome ends, known as telomeric overhangs. in most organisms, the telomeric overhangs consist of g-rich repeats and extend over the c-rich strands at both ends of a chromosome. telomeric overhangs are necessary during telomere replication because they provide a substrate for the rna moiety of the telomerase complex. formation of telomeric overhangs is closely linked to dna replication, occurs independently of telomerase action and is regulated by evolutionary conserved mechanisms. in s. cerevisiae, various exonucleolytic activities are involved in the formation of telomeric ssdna, including the mrx complex and the 5’–3’ double-strand-specific exonuclease exo. the nuclease activity of these enzymes towards chromosome ends is inhibited by telomeric capping factors, such as rap rif and rif that block mrx access, and the cst and ku complexes, which protect telomeres from exo1-mediated degradation. defects in telomere capping that can impact on the activity of exonucleases towards telomere ends lead to various problems, including premature senescence, cell-cycle arrest and accumulation of telomeric ssdna. hence, complete understanding of the factors and mechanisms that control telomere-end processing is important for determining how telomeres maintain their structure and function. in this study, we sought to identify genetic interactions between yeast lincrnas and protein-coding genes in order to evaluate their functional relationships using synthetic genetic array technology. as proof of principle, we initially demonstrated that the gi network of the lincrna tlc is consistent with its function in telomere maintenance. therefore, we next applied the same systematic genetic approach to six other uncharacterised intergenic suts. interestingly, the gi profile of sut connected this lincrna to telomere organisation and follow-up experiments established sut as a novel factor of telomere overhang homeostasis. this study proposes that systematic analysis of gis could unveil the function of lincrnas in s. cerevisiae and other complex organisms.  the gi network of tlc corresponds to its cellular function although genome-wide transcriptional studies have revealed an enormous amount of non-coding rnas that can be synthesised from the s. cerevisiae genome, a relatively small number of these lncrnas have been functionally characterised. we reasoned that we could obtain insights about the function of yeast lincrnas by comprehensively mapping their gis through an approach that has been previously applied for protein-coding genes. therefore, we employed the sga methodology to construct double mutants in which the deletion of an intergenic sut is systematically combined with individual deletions of non-essential genes in budding yeast. gis are scored in double mutants that show significant deviation in fitness compared to the growth of the corresponding single gene deletion strains generated during the control sga screen. specifically, a negative gi refers to a more severe fitness defect in the double mutant compared to the corresponding single gene deletion mutants, while a positive gi corresponds to growth with a less severe fitness defect in the double mutant in comparison to the single deletion mutants. the precise stepwise procedure used for the sga screens and the deviations from a conventional approach are shown in fig. 1a.fig. tlc validates the sga approach for interrogating the function of lincrnas. a schematic illustration of the sga screening procedure used for studying lincrnas. a query strain carrying deletion of a lincrna is mated to an array of single knockout strains, each lacking a non-essential protein-coding gene. following several selection steps, double mutant strains are isolated and their growth fitness is compared to the corresponding single deletion strains derived from a parallel control sga screen. b genetic interactions of tlc associated with the three indicated gene ontology biological processes. gene nodes are coloured according to their assigned go annotation. negative and positive gis that belong to the three go terms are shown. previously established tlc cofactors are highlighted in light grey boxes. c venn diagram showing the significant overlap between genes classified as having a negative gi with tlc and est in this study. the p value was generated using the hypergeometric test to demonstrate proof of concept for the aforementioned rationale, we initially applied the sga procedure to the yeast lincrna tlc which has a well-defined cellular role in telomere maintenance. the sga screen was carried out in duplicate by combining the tlc1Δ strain against an ordered array of approximately viable protein-coding gene deletion strains. a total of ngis and pgis of tlc were identified within the two sga screens. we identified ngis with ku complex components whose synthetic lethal interactions with tlc were previously reported. moreover, tlc showed ngis with genes whose proteins are integral components of the rad52-mediated telomere recombination pathway and the ino chromatin remodelling complex that is also implicated in telomere lengthening via homologous recombination. since pgis are typically detected among components of the same protein complex, we also found that tlc has a positive gi with its co-factors est and est which are subunits of the telomerase complex. to further support the relevance of the identified gis with the function of tlc we then performed gene ontology analysis of our sga data, as an objective metric for deriving functional utility from gi datasets. we chose to perform go enrichment analysis using only ngis, since the sga technique demonstrates higher precision rate on detecting true ngis compared to true pgis and, in addition, ngis frequently occur between genes with overlapping functions. in agreement with the individual gis mentioned above, go analysis of all tlc ngis significantly enriched the biological process terms, telomere organisation and dna recombination. furthermore, tlc ngis significantly enriched the term chromatin remodelling, a cellular activity that has been associated with telomere maintenance. these results show that the sga-derived gis of tlc are consistent with the cellular function of this yeast lincrna.fig. genetic interactions link intergenic suts to diverse biological processes. a heatmap of gene ontology terms enriched by genes whose deletions result in negative genetic interactions. the synthetic genetic array procedure was performed for tlc est and six intergenic suts. the negative genetic interaction from the different sga screens were analysed as described in methods to identify enriched go terms with p ≤ . b and c heatmap of go terms enriched by genes classified as having a ngi with tlc, sut or their corresponding flanking genes. the ngis of the genes flanking the tested lincrnas were extracted from the drygin database and szappanos et al. it was previously demonstrated that genes with highly similar gi profiles have strong functional relationship to the extent that they can be physical partners within the same complex. hence, we hypothesised that tlc should have a gi profile that is highly correlated to the profile of other components of the telomerase complex. to test this hypothesis and support the validity of our tlc sga screens, we performed an independent sga screen for the protein-coding gene est which encodes a subunit of the telomerase complex. we identified ngis for est of which interactions were common with tlc ngis, indicating that their gi profiles are highly related. the shared gis between est and tlc enrich for the go terms telomere organisation and dna recombination that are related to their cellular function. to further examine the validity of our sga screens, we also compared our identified tlc gis to est gis reported in previous studies. notably, about 40% of tlc ngis identified in this study were also identified as est negative gis by two independent sga studies, and once again, the common ngis between tlc and est enrich the go terms telomere organisation and dna recombination. overall, these findings demonstrate that sga-derived gis can illuminate the biological role of tlc and support the utility of this approach in identifying the function of other uncharacterised yeast lincrnas. sga analysis implicates intergenic suts in diverse cellular processes the s. cerevisiae genome encodes approximately suts, of which are intergenic since they do not overlap any other known genomic features. we applied the sga procedure to six intergenic suts that differ in size, distance from adjacent genes and level of expression. the sga screen for each deleted lincrna was paired with a control sga screen to minimise the ‘batch effect’. we identified in total negative gis and positive gis for all six tested suts. we subsequently analysed the ngis of each sut for the enrichment of go biological process terms as described for tlc above. notably, all tested lincrnas enriched at least one go term with the exception of sut which did not highlight any terms despite displaying ngis. it is possible that this lincrna is involved in multiple processes that are diluted among the identified gis. the other five suts exhibited ngis that result in distinct go enrichment profiles. in particular, the sga screen using the sut457Δ strain enriched for the two go terms, telomere organisation and not-yet-annotated. the latter term consists of functionally uncharacterised genes and therefore does not provide any insights into the function of this lincrna. furthermore, sut also showed a specific profile since its screen enriched go terms associated with vesicle function. on the other hand, the other three lincrnas, sut sut and sut exhibited go profiles that are more diverse in function. sut and sut sga screens each enriched seven different biological processes, while sut showed the most varied profile with enrichment of ten different go terms. the heterogeneous go term profiles suggest that the latter three suts are pleiotropic or that the enriched cellular processes are interconnected. altogether, these results indicate that, under physiological conditions, intergenic suts are implicated in a broad spectrum of biological processes that are important for normal cell growth. sut and sut exhibit distinct gi profiles in comparison to their adjacent genes previously characterised suts and other yeast non-coding rnas have been linked to transcriptional regulation of their neighbouring genes. therefore, we reasoned that sga analysis of lincrnas, which control their cognate genes, will lead to the discovery of gis which would be similar to those identified for their neighbouring genes. to compare the gi networks of the examined intergenic suts with those of their neighbouring genes, we exploited the gi data of protein-coding genes available on the drygin dataset and those generated by szappanos et al., which are compatible with the sga approach used in this study. we first compared the go enrichment profiles of tlc with its upstream and downstream gene, pdx and csg respectively, since tlc is not linked to the transcriptional control of its neighbours. as predicted, the gis of tlc enrich go terms that are distinct to those enriched by the gis of its flanking genes. similarly to tlc two other lincrnas, sut and sut exhibit go enrichment profiles that are completely different from those of their upstream and downstream neighbouring genes. in contrast, sut sut and sut whose gis enrich diverse biological processes, display go profiles that partially overlap those of their adjacent genes. these findings suggest that sut sut and sut might have functions that are linked to the expression of their neighbouring genes, while sut and sut have roles that are independent to the function of their adjacent genes. deletion of sut accelerates senescence in telomerase-deficient cells sut showed a very specific go enrichment profile that was distinct from its flanking genes and, therefore, we decided to further characterise its cellular role. initially, we verified by northern blot analysis the loss of sut transcript in the sut457Δ strain which was processed in the sga screens. then, we examined whether construction of the sut457Δ strain genetically perturbs the expression of its neighbouring genes. quantitative rt-pcr analysis showed that in sut457Δ cells the expression of sut457-adjacent genes syp snr and rps14a remains unaffected compared to a wild-type control strain. this result demonstrates that construction of sut deletion does not lead to the previously reported neighbouring gene effect and, hence, it confirms that the gi network of sut457Δ is directly associated with sut itself and not with its adjacent genes.fig. deletion of sut accelerates senescence in telomerase-deficient cells. a schematic representation of the sut locus on chromosome iii. arrows indicate direction of transcription. b expression levels of syp sut snr and rps14a determined by qrt-pcr using total rna extracted from isogenic wild-type and sut457Δ strains. the expression levels of each gene were normalised to the expression of act error bars represent standard error of the mean, resulting from three independent replicates. *** p < ; n/s, not significant. c genetic interactions of sut associated with the gene ontology term telomere organisation. gene names in bold represent negative genetic interactions while gene names in regular font correspond to positive genetic interactions. d telomere length analysis of genomic dna isolated from the indicated wild-type and mutant strains at passage the extracted dna was fragmented with xhoi and subjected to denatured southern blotting using a biotinylated probe against telomeric repeats. e senescence assays performed using liquid cultures of the indicated isogenic wild-type and mutant strains. the strains were generated through tetrad dissection of the heterozygous diploid double mutant sut457/sut457Δtlc1/tlc1Δ. this plot represents one of three independent tetrads examined. f telomere length analysis performed as in using genomic dna isolated from the indicated wild-type and mutant strains during passages and 8 sut genetically interacts with out of telomere organisation genes found in the sga deletion collection. these genes are associated with roles in supporting telomerase function, modulating subtelomeric chromatin changes, regulating telomere-end processing, and controlling telomere maintenance via recombination. the genetic link between sut and telomere organisation initially prompted us to determine if loss of this lincrna leads to changes in telomere length. to do this, we used denaturing southern analysis and found that deletion of sut alone does not affect telomere length compared to an isogenic wild-type strain. then, we examined whether sut457Δ, in combination with deletion of any of its genetic interactors, affects telomere length. as expected, significantly shorter telomeres were detected in the yku70Δ and mre11Δ single mutants compared to wild-type cells, but telomere length was not further affected in the yku70Δsut457Δ or mre11Δsut457Δ double mutants. consistently, sut457Δ, in combination with deletion of the remaining telomere-related genetic interactors, does not have a significant change in telomere length compared to its isogenic single deletion mutants. based on the fact that some of the telomere-associated gis of sut are known to affect entry into senescence in telomerase-deficient cells, we next sought to determine if deletion of sut influences the rate of senescence in tlc null cells. therefore, we performed liquid senescence assays using the four isogenic strains, namely wild-type, sut457Δ, tlc1Δ and the double mutant sut457Δtlc1Δ. consistent with the fact that single deletion of sut had wild-type telomeric length, we observed that sut457Δ and wild-type strains had a very similar growth profile in senescence assays. however, we found that the double mutant sut457Δtlc1Δ had an accelerated entry into senescence compared to the tlc1Δ single mutant. interestingly, this faster induction of senescence correlates with the appearance of shorter telomeres in the sut457Δtlc1Δ double deletion strain compared to the tlc1Δ single mutant. moreover, deletion of sut did not affect the generation of type ii survivors in strains lacking telomerase. altogether, these findings link the function of sut to telomere organisation and specifically show that sut participates within a telomerase-independent pathway. loss of sut leads to accumulation of telomeric single-stranded dna it has been previously reported that faster senescence in telomerase-negative cells can be attributed to abnormal accumulation of telomeric ssdna. thus, we hypothesised that deletion of sut may lead to the accumulation of ssdna at telomeres. this hypothesis was further supported by the sga findings above, which show that sut genetically interacts with the genes yku mre tgs cgi hsc and gbp that encode for factors implicated in telomere-end protection and in the formation of telomeric ssdna overhang. to examine this hypothesis, we extracted genomic dna from isogenic wild-type and sut457Δ cells at an early passage and performed native southern blotting using a c-rich oligonucleotide probe. in addition, we extracted genomic dna from a later passage to mimic the subculturing of cells resulting from the serial pinning steps performed during the sga procedure. absence of sut led to an increase in the signal of telomeric ssdna compared to wild-type cells in passage, which became even more intense after subculturing yeast cells for five passages. in order to verify that the detected ssdna signal is due to an increase in 3’ terminal overhang and not due to accumulation of internal dna replication intermediates, we treated the native dna with bacterial exonuclease i that can only degrade terminal ssdna in a 3’ to 5’ direction. the single stranded dna that accumulated in sut457Δ mutant was sensitive to bacterial exonuclease i digestion, indicating that the detected ssdna indeed corresponds to telomeric 3’ overhang. we also validated the increase in telomeric overhang by comparing the levels of ssdna among different sut457Δ mutant clones and their corresponding wild-type strains. as above, we observed statistically significant accumulation of telomeric ssdna in sut457Δ strains at an early passage which became even more substantial at a later passage.fig. loss of sut leads to exo1-dependent accumulation of telomeric ssdna. a analysis of telomeric ssdna overhangs in isogenic wild-type and sut457Δ strains. yeast colonies were restreaked on agar plates and genomic dna was extracted at passages and, fragmented with xhoi and subjected to native southern blotting using a biotinylated probe against telomeric repeats. the southern blot was then treated with  n naoh and the denatured dna was re-probed to monitor equal loading. b accumulation of telomeric ssdna in eleven sut457Δ strains compared to their respective wild-type strains during passages and. the ssdna signal for each wild-type and mutant strain detected in the native southern blot was normalised to the corresponding signal in the denatured southern blot. error bars represent standard error of the mean. * p < ; ** p < ; n/s, not significant. c cell growth was monitored for the indicated isogenic strains at passages and d analysis of telomeric ssdna levels at passage of wild-type, sut457Δ, exo1Δ and exo1Δsut457Δ isogenic strains generated through dissection of the heterozygous diploid double mutant exo1/exo1Δsut457/sut457Δ. southern blotting was performed as in above. e telomeric ssdna levels analysed at passage in the indicated isogenic wild-type and mutant strains generated from three independent tetrads. the quantification of the ssdna signal for each wild-type and mutant strain was performed as in above. error bars represent standard error of the mean. ** p < ; n/s, not significant. the statistical analysis was performed using one-way anova accumulation of telomeric ssdna has been proposed to be a signal for cell cycle arrest. however, we did not observe a growth reduction in the sut457Δ mutant strain compared to wild-type cells. it was previously demonstrated that growth arrest associated with telomeric overhang accumulation is masked by telomeric ssdna binding proteins like gbp. interestingly, gbp was identified as a sut ngi and, therefore, we next examined how lack of gbp can affect the growth of sut457Δ cells. we observed a strong growth arrest of gbp2Δsut457Δ cells at passage, which coincides with the robust accumulation of telomeric ssdna. altogether, these data suggest that sut controls the levels of telomeric ssdna overhang in order to maintain proper cellular growth. exo nuclease is required for the accumulation of telomeric ssdna in sut457Δ cells the 5’–3’ exonuclease exo plays a key role in telomere g-rich overhang formation in yeast by mediating c-rich strand degradation. the action of exo is blocked by the ku complex since lack of yku leads to unprotected telomere ends and results in exo1-dependent accumulation of telomeric ssdna. because sut genetically interacts with yku, we hypothesised that exo activity may be responsible for the accumulation of telomeric overhang in sut457Δ cells. to test this hypothesis, we wanted to generate an exo1Δsut457Δ double mutant strain by crossing the exo1Δ and sut457Δ single mutants, but we realised that exo was not properly deleted in the exo1Δ strain found in the sga knockout library. therefore, we constructed a new exo1Δ deletion strain, mated it to the sut457Δ mutant and generated isogenic haploid strains that were analysed for telomeric ssdna levels. consistent with the above findings, we detected a strong signal in sut457Δ corresponding to the accumulation of telomeric ssdna after subculturing yeast for passages. notably, the telomeric overhang signal in the exo1Δsut457Δ double mutant cells is reduced back to wild-type levels. this result was verified by quantifying the levels of telomeric ssdna in isogenic mutant strains generated from three independent tetrads. furthermore, deletion of exo in gbp2Δsut457Δ cells rescued their growth arrest at passage suggesting that exo1Δ suppresses the effect of sut457Δ. altogether, these findings show that sut functions within an exo1-dependent pathway to affect telomeric overhang accumulation. the lincrna sut acts in trans to regulate the levels of telomeric ssdna the gi profile of sut was distinct from that of its adjacent genes and this is also consistent with the fact that loss of sut does not affect the expression of its neighbours. therefore, we hypothesised that, unlike most characterised yeast lncrnas, which control the expression of their cognate genes, sut may function in trans distant from its locus of synthesis. to address this hypothesis, we examined whether ectopic expression of sut could rescue the phenotype of telomeric overhang accumulation observed in sut457Δ cells. for this purpose, we deleted sut from its endogenous locus on chromosome iii and re-introduced it on chromosome v under the control of the ura promoter. we also constructed another strain in which sut was ectopically expressed at the ura locus in wild-type cells to serve as an additional control. native southern blot analysis indicated that deletion of sut457Δ from its endogenous locus results in robust increase in telomeric ssdna at passage as previously shown, but concomitant ectopic expression of sut reduces the telomeric overhang signal to corresponding wild-type levels. this result was verified by quantifying the levels of telomeric ssdna in five independent constructed strains. collectively, these findings demonstrate that sut can control the levels of telomeric ssdna overhang regardless of its genomic location, indicating that this lincrna functions in trans.fig. sut regulates the levels of telomeric ssdna by acting in trans. a schematic illustration indicating the presence of endogenous and ectopic sut in wild-type and sut457Δ strains. the level of sut expression for each constructed strain is indicated on the right: +, +++, and –. arrows indicate the direction of sut transcription at each locus. b analysis of telomeric ssdna levels in wild-type, wild-type, sut457Δ, and sut457Δ strains at passage genomic dna was extracted, fragmented with xhoi and subjected to native southern blotting using a biotinylated probe against telomeric repeats. the southern blot was then treated with  n naoh and the denatured dna was re-probed to monitor equal loading. c telomeric ssdna levels were analysed in five independent clones of the indicated wild-type and mutant strains at passage the ssdna signal for each strain detected in the native southern blot was normalised to the corresponding signal in the denatured southern blot. error bars represent standard error of the mean. * p < ; n/s, not significant. statistical analysis was performed using one-way anova discussion despite the plethora of long non-coding rnas identified by genome-wide approaches, rigorous genetic studies that could aid in the functional characterisation of individual lncrnas are missing. here, we took advantage of the genetic tractability of yeast and employed a high-throughput sga methodology to systematically catalogue the gis of six intergenic suts. initially, we verified the utility of the approach by demonstrating that gis identified for the telomerase rna tlc enrich go terms corresponding to its known cellular function. subsequent sga screens and analysis of gi profiles implicated the six tested intergenic suts in diverse biological processes. one of these lincrnas, sut exhibited gis that enriched a definitive go annotation linking it to telomere biology. further functional characterisation unveiled the role of sut in preventing the accumulation of telomeric ssdna. moreover, phenotypic rescue experiments showed that sut acts in trans to maintain physiological levels of telomeric ssdna. our work reveals that systematic mapping of the gis of lncrnas could associate these molecules with specific biological processes and pinpoint their individual functions. in yeast, the function of identified lncrnas has been mainly linked to the regulation of gene expression. most lncrnas characterised so far regulate their cognate genes either through transcriptional interference or by modulating their local chromatin structure. however, emerging evidence suggest that lncrnas could have roles beyond gene regulation. the go enrichment profiles of the tested intergenic suts have linked them to nuclear functions such as dna repair, meiosis and telomere organisation but also to cytoplasmic processes including membrane fusions and vesicle formation, for example, in the case of sut. the latter observation is consistent with the fact that a large number of suts are transported to the cytoplasm and have been proposed to represent functional transcripts within this cell compartment. moreover, human lncrnas are co-expressed with cytoplasmic membrane proteins and are present within extracellular vesicles. therefore, future in-depth characterisation of lincrnas, like sut could elucidate the role of these molecules in cytoplasmic processes such as vesicle biogenesis and trafficking. only rare examples of trans-acting yeast lncrnas have been reported to date. specifically, the pho antisense lncrnas and a cryptic unstable transcript associated with ty retrotransposons control transcriptional silencing of their target genes in trans. we show here that gi profiling could also provide insights about the mode of action of lincrnas in addition to inferring their biological function. this is achieved by comparing the gi profiles of lincrnas with those of their adjacent protein-coding genes. for example, tlc has a gi network that completely differs from that of its upstream and downstream genes which is in line with the fact that tlc has a function unrelated to its neighbours and acts in trans at chromosome ends. similarly, sut and sut have distinct go profiles compared to their neighbouring genes, suggesting that these suts may also work in trans. accordingly, sut deletion does not affect the expression of its neighbouring genes and regulates a telomeric process occurring distal from its transcriptional site. however, there is still a possibility that the sut locus is proximal to a gene involved in telomere-end processing within the three-dimensional architecture of the genome. hence, in such a scenario, sut would have a role in telomere-end processing by controlling its spatially proximal gene. nevertheless, the phenotypic rescue obtained by expressing sut from a different chromosome makes this scenario rather unlikely. three of the other intergenic suts tested exhibit gis with go enrichment profiles that overlap those of their flanking genes, indicating that these lincrnas may be involved in the regulation of their nearby genes in cis. of course, we cannot eliminate, at this point, the likelihood that construction of these three sut deletions leads to a neighbouring gene effect, which might be responsible for the observed overlap among enriched go profiles. we note that two of the six tested intergenic suts exhibit gi profiles consistent with a trans-acting role and, therefore, in contrast to current evidence in the literature, we anticipate that a considerable number of budding yeast lincrnas would function at sites distal from their locus of synthesis. lack of sut exhibited accelerated senescence in telomerase-negative cells associated with enhanced telomere shortening and an accumulation in telomeric ssdna overhang. interestingly, analogous phenotypes have been reported for mutations of factors involved in telomere-end protection. consistent with the above observations, sut shows a positive gi with the telomere-capping factor yku, implying that these two molecules affect the same molecular process. notably, yku binds to and protects telomeres from exo nucleolytic processing, thus preventing the accumulation of telomeric ssdna overhang. our data also show that the exo nuclease is required for the increase in telomeric ssdna detected in the absence of sut. furthermore, in agreement with the fact that exo1-dependent accumulation of telomeric ssdna induces cell-cycle arrest, we show that loss of the telomeric ssdna masking protein gbp in sut457Δ cells results in growth inhibition after subculturing and, importantly, this growth arrest is rescued by deletion of exo. based on the above evidence, we speculate that sut functions in a pathway that protects telomere-ends from nucleolytic processing in order to regulate the levels of telomeric ssdna overhangs. interestingly, the telomere-associated lncrna telomeric repeat-containing rna has also been implicated in this pathway but instead facilitates the nuclease activity of exo at chromosome ends. the primary nucleotide sequence of sut does not display significant sequence similarity with any other regions within the s. cerevisiae genome and lacks conservation even amongst closely related yeast species. nevertheless, considering its unveiled role in the fundamental and conserved cellular process of telomere overhang homeostasis, we believe that functional analogues of sut may exist in other eukaryotes. likewise, tlc is a lncrna whose primary sequence is not evolutionarily conserved but which has functional analogues in other organisms.  transcriptome studies have enabled the discovery of a large number of lncrnas in various species. however, it remains unclear how many of these lncrnas serve a biological function or are a mere result of transcriptional noise. we show here that systematic analysis of pairwise gis can provide insights about the function of individual lncrnas. the functional maps obtained by gis could complement the information derived by other high-throughput approaches to help unravel the biological significance of lncrnas. although the present study focuses on stable lincrnas, future work could also analyse gis for yeast antisense lncrnas since methodologies are being developed that abrogate the transcription of a specific non-coding rna without disrupting the expression of its associated sense mrna. furthermore, global mapping of gis for lncrnas can be conceivably extended to complex organisms because recent advances in rnai and crispr/cas technologies allow for efficient pairwise gene disruptions in human cells. such analyses may unveil interspecies conservation of gi maps that could shed light on the evolution and functional conservation of lncrnas. 
overall, our work proposes that systematic application of this genetic approach could determine the functional significance of individual lncrnas in yeast and other complex organisms. we subsequently performed sga screens on a set of uncharacterised lincrnas and uncover their connection to diverse cellular processes. we first validate this approach by demonstrating that the telomerase rna tlc displays a gi network that corresponds to its well-described function in telomere length maintenance. however, only a few lncrnas have been ascribed a cellular role thus far, with most regulating the expression of adjacent genes. here, we employ synthetic genetic array in saccharomyces cerevisiae to identify gis between long intergenic non-coding rnas and protein-coding genes. transcriptome studies have revealed that many eukaryotic genomes are pervasively transcribed producing numerous long non-coding rnas. long intergenic non-coding rnasstable unannotated transcriptssynthetic genetic arraygenetic interactionstelomerehttp://dx.doi.org/ /501100000781european research council260797kirmizis antonis issue-copyright-statement© the author 2016 furthermore, the gi profile of sut is distinct from that of its neighbouring genes suggesting a function independent to its genomic location. one of these lincrnas, sut exhibits a gi profile associating it to telomere organisation and we consistently demonstrate that sut is required for telomeric overhang homeostasis through an exo1-dependent pathway. accordingly, we show that ectopic expression of this lincrna suppresses telomeric overhang accumulation in sut457Δ cells assigning a trans-acting role for sut in telomere biology. therefore, the function of lncrnas could be illuminated through systematic analysis of their synthetic genetic interactions.
 transcriptome studies have revealed that many eukaryotic genomes are pervasively transcribed producing numerous long non-coding rnas. however, only a few lncrnas have been ascribed a cellular role thus far, with most regulating the expression of adjacent genes. even less lncrnas have been annotated as essential hence implying that the majority may be functionally redundant. therefore, the function of lncrnas could be illuminated through systematic analysis of their synthetic genetic interactions.  here, we employ synthetic genetic array in saccharomyces cerevisiae to identify gis between long intergenic non-coding rnas and protein-coding genes. we first validate this approach by demonstrating that the telomerase rna tlc displays a gi network that corresponds to its well-described function in telomere length maintenance. we subsequently performed sga screens on a set of uncharacterised lincrnas and uncover their connection to diverse cellular processes. one of these lincrnas, sut exhibits a gi profile associating it to telomere organisation and we consistently demonstrate that sut is required for telomeric overhang homeostasis through an exo1-dependent pathway. furthermore, the gi profile of sut is distinct from that of its neighbouring genes suggesting a function independent to its genomic location. accordingly, we show that ectopic expression of this lincrna suppresses telomeric overhang accumulation in sut457Δ cells assigning a trans-acting role for sut in telomere biology.  overall, our work proposes that systematic application of this genetic approach could determine the functional significance of individual lncrnas in yeast and other complex organisms. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords long intergenic non-coding rnasstable unannotated transcriptssynthetic genetic arraygenetic interactionstelomerehttp://dx.doi.org/ /501100000781european research council260797kirmizis antonis issue-copyright-statement© the author 2016 