Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Meiotic Recombination Hotspots of Fission Yeast Are Directed to Loci that Express Non-Coding RNA

  • Wayne P. Wahls ,

    wahlswaynep@uams.edu

    Affiliation Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America

  • Eric R. Siegel,

    Affiliation Department of Biostatistics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America

  • Mari K. Davidson

    Affiliation Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America

Meiotic Recombination Hotspots of Fission Yeast Are Directed to Loci that Express Non-Coding RNA

  • Wayne P. Wahls, 
  • Eric R. Siegel, 
  • Mari K. Davidson
PLOS
x

Abstract

Background

Polyadenylated, mRNA-like transcripts with no coding potential are abundant in eukaryotes, but the functions of these long non-coding RNAs (ncRNAs) are enigmatic. In meiosis, Rec12 (Spo11) catalyzes the formation of dsDNA breaks (DSBs) that initiate homologous recombination. Most meiotic recombination is positioned at hotspots, but knowledge of the mechanisms is nebulous. In the fission yeast genome DSBs are located within 194 prominent peaks separated on average by 65-kbp intervals of DNA that are largely free of DSBs.

Methodology/Principal Findings

We compared the genome-wide distribution of DSB peaks to that of polyadenylated ncRNA molecules of the prl class. DSB peaks map to ncRNA loci that may be situated within ORFs, near the boundaries of ORFs and intergenic regions, or most often within intergenic regions. Unconditional statistical tests revealed that this colocalization is non-random and robust (P≤5.5×10−8). Furthermore, we tested and rejected the hypothesis that the ncRNA loci and DSB peaks localize preferentially, but independently, to a third entity on the chromosomes.

Conclusions/Significance

Meiotic DSB hotspots are directed to loci that express polyadenylated ncRNAs. This reveals an unexpected, possibly unitary mechanism for what directs meiotic recombination to hotspots. It also reveals a likely biological function for enigmatic ncRNAs. We propose specific mechanisms by which ncRNA molecules, or some aspect of RNA metabolism associated with ncRNA loci, help to position recombination protein complexes at DSB hotspots within chromosomes.

Introduction

Over the past decade it has become clear that non-coding RNA (ncRNA) molecules have a major role in a vast array of diverse cellular processes. Small (∼20–30 nt) ncRNAs such as siRNA and miRNA can trigger the formation of euchromatin or heterochromatin, affect positively or negatively transcription, induce the deadenylation of mRNA, trigger the targeted degradation of mRNA, and regulate positively or negatively the translation of functional mRNAs (reviewed by [1], [2]). Nucleotide sequence complementarity between the small ncRNAs and target molecules serves to guide various protein complexes to the appropriate targets within mRNA (e.g., for cleavage by Argonaute) [3] or within DNA of chromosomes (e.g., for heterochromatinization by RITS) [4].

Another class of ncRNA molecules, the larger mRNA-like transcripts with little or no coding potential, are ubiquitous in eukaryotes ranging from fission yeast to humans [5], [6]. While the precise values are unknown and vary from organism to organism, the total number of polyadenylated ncRNAs may exceed the number of protein-coding mRNAs. With a few exceptions (e.g., [7][10]) the function of these long ncRNAs is completely obscure, but it seems almost certain that they (like small ncRNAs) will be found to play important roles in the cell [1], [2]. We report here an unexpected, robust connection between such ncRNAs and meiotic chromosome dynamics.

In meiosis, a combination of crossover recombination structures (chiasmata) and sister chromatid cohesion distal to chiasmata help to align homologous chromosome pairs and ensure their proper segregation in the first meiotic division (Figure 1) [11]. Consequently, meiotic recombination is not distributed randomly, but is tightly regulated to ensure that each chromosome pair receives at least one chiasma. Furthermore, recombination is positioned preferentially at hotspots along each chromosome, but current knowledge of the mechanisms for this clustering is knowledge of mechanisms for this clustering is nebulous (reviewed by [12][14]). The DNA binding sites for some transcription factors are hotspots [15][22], but there is no obvious DNA sequence preference (i.e., specific consensus sequence) for hotspots across the genome [23], [24]. The relative accessibility of DNA in regions of “open” chromatin associated with transcription might facilitate the entry of meiotic recombination enzymes [16], [25][28], but open chromatin is insufficient to promote recombination [29] and some hotspots lack open chromatin [30], [31]. The binding of certain transcription factors [16], [22], [32], chromatin remodeling by transcription factors [25], [33], and transcription levels [19], [34], [35] either influence or are essential for local hotspot activity. Despite all of these seemingly clear connections to transcription, and paradoxically, hotspots in diverse organisms tend to cluster preferentially in non-coding regions [23], [24], [30]. It is therefore likely that additional, yet-unidentified factors help to regulate where meiotic recombination occurs.

thumbnail
Figure 1. Hallmarks of meiosis.

After DNA replication, homologous chromosomes (light and dark) pair and undergo a high rate of recombination. Recombination events are not distributed randomly, but cluster at hotspots. Structures created by crossover recombination (chiasmata) and sister chromatid cohesion (stars) facilitate the alignment and segregation of homologs in the first meiotic division. The second meiotic division is similar to mitosis, where sister chromatids segregate to opposite poles.

https://doi.org/10.1371/journal.pone.0002887.g001

The meiotic recombination protein Spo11 (Rec12 in fission yeast) is a conserved, topoisomerase II-like enzyme which introduces dsDNA breaks (DSBs) that initiate recombination [36]. These meiotic DSBs have been well characterized in two highly-diverged [37] organisms, budding yeast and fission yeast, and likely reflect a common mechanism for the initiation of recombination in all eukaryotes (reviewed by [14]). A recent analysis of DNA tiling microarrays revealed the genome-wide distribution of DSBs in fission yeast [23]. Most of the DSBs are clustered within 194 prominent peaks that are spaced on average about 65 kbp apart, and between the peaks there are few, if any, detectable DSBs. The DSB clusters are found in both coding regions (i.e., genes) and in intergenic regions (IGRs), but they localize preferentially to large IGRs. Other than this general bias towards IGRs, no discrete features which might be responsible for DSB hotspot clustering were identified [23]. We report here that the DSB hotspots are directed preferentially to loci that express polyadenylated ncRNAs. We propose specific mechanisms by which ncRNA molecules, or features of ncRNA loci, help to regulate the positioning of meiotic recombination.

Results

Loci that express polyadenylated, ncRNAs are embedded within DSB peaks

Our long-term interest is to define how meiotic homologous recombination becomes localized at hotspots. We therefore examined the genomic DNA sequences surrounding meiotic DSB peaks of fission yeast, and we discovered that several of the peaks encompass DNA sequences which express ncRNA molecules. For example, a prominent DSB peak within the rec7 gene encompasses three polyadenylated, transcript from opposite strand RNA molecules, tos1, tos2, and tos3 (Figure 2A). These non-coding tos transcripts are induced only in meiosis [38] and are therefore present when Rec12 (Spo11) catalyses the formation of DSBs. Similarly, some prominent DSB peaks contain prl transcripts (polyA-bearing RNA without long open reading frames [5]) (e.g., Figure 2B). Such non-coding prl transcripts are expressed in meiosis, are found within both coding regions and IGRs but localize preferentially to large IGRs, and some of them are spliced [5], [39]. In other words, the distribution and developmental regulation of some polyadenylated, ncRNA molecules seem to coincide with those of prominent DSB peaks.

thumbnail
Figure 2. Colocalization of DSB hotspots with ncRNA.

The frequencies of DSBs for each oligonucleotide tile (circles) and DSB peaks (curves) were plotted as a function of distance and are shown relative to the positions of protein-coding ORFs (boxes) and long ncRNAs (tos and prl, arrowhead indicates poly-A tail). The ncRNAs can be found within “weak” (C, I) and “prominent” (A, B, D–H) DSB peaks. Peaks and ncRNAs can map together within ORFs (A), near ORF-IGR boundaries (D, E), or within IGRs (B, C, F–I). The DSB-associated, polyadenylated ncRNAs may be spliced (H, I) or not (A–G).

https://doi.org/10.1371/journal.pone.0002887.g002

Genome-wide analyses of association between prl loci and DSB peaks

The fission yeast genome contains about 5,000 coding genes [40]. Among 987 cDNA clones of a random library derived from cells in mitosis and meiosis, 68 correspond to polyadenylated, ncRNAs of the prl class [5]. This sample is of sufficient size and complexity to be representative of the distribution and abundance of prl molecules expressed from the genome. We therefore compared the genome-wide distribution of DSB peaks obtained from microarray analysis [23] to that of genomic DNA sequences from which the representative prl molecules are transcribed [5] (Table 1). Since a subset of the DSB peaks were classified previously as being “prominent” (as opposed to “weak”) [23], we analyzed both the prominent peaks alone and all peaks together (prominent plus weak).

thumbnail
Table 1. Genome-wide distribution of representative prl loci and their proximity to DSB peaks.

https://doi.org/10.1371/journal.pone.0002887.t001

The average distance between prominent DSB peaks is 65 kbp and the average distance between the representative prl loci is 185 kbp. One might expect these prl loci to map, on average, about 33 kbp away from DSB peaks. However, the distribution of distances between prl loci and their neighboring DSB peaks is skewed markedly towards a much shorter distance (Figure 3A–B). Furthermore, there is an unexpectedly high frequency of perfect colocalization (Figure 2, Table 1): Of the 68 ncRNA molecules analyzed, 18 (26.5%) map entirely within prominent DSB peaks. Six additional ncRNAs (8.8%) map entirely within weak DSB peaks. Fourteen more (20.5%) fall on or near the flanks of DSB peaks (within 5 kbp). In toto, about 56% of the representative ncRNA loci are associated with DSB peaks by these criteria. To analyze these data further, we calculated the unconditional genome-wide probabilities of perfect colocalization, which are the most conservative and rigorous criteria possible.

thumbnail
Figure 3. Non-random association of ncRNA with DSB hotspots.

(A) Relative proximity of prl transcripts to prominent DSB peaks. Shown are the binned distributions of distances between each prl locus and the nearest DSB peak. Under the null hypothesis of no association, the mean and mode would fall in the fourth bin. (B) Relative proximity of prl transcripts to all (prominent and weak) DSB peaks, analyzed as in panel “A”. Under the null hypothesis, the mean and mode would fall in the third bin. (C) Non-random colocalization of DSB peaks with ncRNA of the prl class. Plot shows the expected (exp) and observed (obs) values for the localization of prl molecules entirely within DSB peaks.

https://doi.org/10.1371/journal.pone.0002887.g003

The 194 prominent DSB peaks encompass about 700 kbp (5.6%) of the genome sequence and those peaks are separated by regions essentially devoid of DSBs [23]. One can view the ncRNA molecules as having a binomial distribution with respect to landing successfully (yes or no) within a DSB peak. We therefore analyzed the data using the exact binomial test of a single proportion. The probability of success under the null hypothesis of random placement anywhere within the genome would be 5.6% per molecule. This yields an expected value of 3.8 out of 68 prl molecules falling within a prominent DSB peak, and 96.4% confidence that the number of molecules within a prominent peak should be seven or less. The probability of observing 18 or more prl molecules out of 68 landing within a prominent DSB peak by chance is low (P = 2.5×10−8) (Figure 3C). We conclude that there is a positive, non-random association between loci which express the prl class of ncRNA molecules and the positions of prominent DSB clusters that initiate meiotic recombination.

The preceding analyses considered the 194 prominent DSB peaks that collectively contain most of the meiotic DSBs catalyzed by Rec12 (Spo11). However, there are an additional 159 “weak” DSB peaks that encompass less than 5% of the genome and whose aggregate area integrals contribute 12% of the total DSBs measured across the genome [23]. Within these weak peaks we found six additional ncRNA transcripts of the prl data set (e.g., Figure 2C, I). Therefore 35.3% (24 of 68) of the prl molecules fall entirely within a DSB peak (weak or prominent). The complete set of all detectable DSB peaks (weak and prominent) map to about 10.6% of the genome sequence [23]. Under a null-hypothesis success probability of 10.6% per molecule, one would expect 7.2 of the 68 prl molecules to fall within a peak by chance, and have 97.4% confidence that the number falling within a peak would be 12 or less. The probability of observing 24 or more occurrences out of 68 is low (P = 5.5×10−8) (Figure 3C). We conclude that there is a positive, non-random association between prl loci and DSB peaks for the entire contingent of all known DSB peaks (prominent and weak) across the genome.

Test of an alternative hypothesis: Independent localization to IGRs?

About 81% of the prominent DSB peaks, 61% of the weak DSB peaks, and 87% of the representative prl loci are within IGRs [5], [23]. This raises the possibility that the apparent non-random associations noted above arise simply from prl loci and DSB peaks localizing preferentially, but independently, to IGRs. To explore this possibility further, we restricted the analysis to the 40% of the fission yeast genome comprised of IGRs, and we computed the corresponding conditional probabilities under the binomial distribution and null hypothesis of no association. DSB peaks (prominent and weak) cover 22% of DNA within IGRs, prominent peaks cover 14% of DNA within IGRs, and 59 prl loci are specific to IGRs. Twenty four of the 59 IGR-specific prl loci (41%) occur within a DSB peak (P = 9.6×10−4), 18 of which (31%) occur within a prominent peak (P = 8.6×10−4). We conclude that the intimate association between DSB peaks and prl loci does not arise from independently preferential localization of each to an IGR. Rather, the DSB peaks colocalize specifically with ncRNA loci that may be situated within ORFs, near ORF-IGR boundaries, or most often within IGRs (e.g., Figure 2).

Discussion

It is implausible, statistically, that the association between DSB peaks and ncRNAs of the prl class is coincidental (P≤5.5×10−8, Figure 3C). The strength of this association was documented using the most strict criteria possible (perfect colocalization) and it takes into account all of those instances where DSB peaks and representative ncRNA loci do not colocalize precisely. It applies whether one examines only the prominent DSB peaks or all known DSB peaks. Moreover, we tested and rejected the hypothesis that prl loci and DSB peaks localize preferentially, but independently, to a third entity on the chromosomes. Together these findings support an unambiguous, overall conclusion—meiotic DSB hotspots of fission yeast are directed preferentially to loci that express long, polyadenylated ncRNA molecules of the prl class.

DSB hotspots localize to ncRNA loci far more intimately than to any other known factors

The genome-wide, preferential localization of DSB peaks to representative ncRNA loci is unconditional and robust (P≤5.5×10−8, Figure 3C). For comparison, three other factors are reported to correlate positively with the genome-wide distribution of DSB hotpots, at least under conditional parameters [23], [24]. In both fission yeast and budding yeast, DSB peaks are associated with a slightly elevated G:C content (P>0.05 to P≤0.0001, depending upon window size and the other conditional parameters applied). In both yeasts there is also an association of DSB peaks with those IGRs located 5′ of protein-coding genes that are divergently transcribed (P = 0.001 to P≤0.0001, from conditional assessment of IGRs alone). This correlation breaks down (P>0.05) in fission yeast when the size of the IGR is controlled for; such IGR size controls were not reported for budding yeast. And in fission yeast, the presumptive promoter regions of genes with DSB peaks are enriched for gene ontology (GO) terms associated with “interaction between organisms” or “transcription factor activity” (from conditional assessment of presumtive promoter regions alone, no P values reported). Among these various factors, the presence of loci expressing the polyadenylated prl class of ncRNA is by far the single best predictor of where meiotic DSB peaks localize in the genome (P≤5.5×10−8, Figure 3C).

Transcripts of the prl class are expressed in meiosis (many exclusively in meiosis) [5], [38], [39], so they are present at the developmental stage in which Rec12 (Spo11) catalyzes the formation of DSBs. We therefore propose that ncRNA molecules or loci of the prl class help to activate DSB hotspots. This process may be conserved, because in mice one ncRNA has been shown to map to a well-defined meiotic recombination hotspot [41] and at least one other has been implicated to do so [42].

Minimum and maximum estimates of potency

The vast majority of fission yeast ncRNAs remain undiscovered [5], so the presence and frequency of ncRNA-free DSB peaks is uninformative scientifically. We note, however, that there are enough predicted ncRNA loci [5] to populate each of the known DSB peaks.

About 35% of the representative prl loci are embedded entirely within DSB peaks (Figures 23, Table 1), so if one excludes any possible function at distance the remaining 65% of prl loci would not be sufficient to promote recombination. However, two factors suggest that the fraction of potentially recombinogenic prl loci may be much greater than 35%. First, the distribution of DSB peaks in fission yeast was determined using rad50S strains [23], and in rad50S strains of budding yeast about half of all meiotic DSB peaks escape detection [43], [44]. If this applies to fission yeast, then there would be about twice as many DSB peaks as reported. In that case, somewhere between at least 35% (the current observed value) and approximately 70% (an extrapolated value) of the representative prl loci would fall entirely within DSB peaks. Second, if one allows for possible function at even a very short distance (e.g., ≤5 kbp), then the fraction of prl loci associated with DSB peaks also increases dramatically (by 58%, Table 1). Such possible function at distance is indicated clearly by the data (means and modes, Figure 3A–B). For these reasons, the theoretical maximum value for functional association may approach unity.

Hypothetical mechanisms: Chromatin structure or guide RNA

How might long, polyadenylated ncRNA molecules or loci that express ncRNA molecules help to position the initiation of meiotic recombination catalyzed by Rec12 (Spo11)? We propose two, not mutually exclusive, hypotheses on mechanism.

A chromatin docking-site hypothesis for hotspot meiotic recombination at ncRNA loci.

In fission yeast meiotically induced chromatin remodeling occurs at DSB hotspots [45], [46] and where tested is seemingly required for hotspot activity [25]. Therefore one possibility is that prl-dependent chromatin remodeling, due either to some aspect of transcription at prl loci or mediated by the ncRNA molecules themselves, creates a preferential site for the nucleation of recombination protein complexes. This hypothesis fits comfortably within the prevailing orthodoxy, which posits that the relative accessibility of DNA in regions of “open” chromatin has a role in hotspot activity (reviewed by [12], [14], [47], [48]).

A guide RNA hypothesis for hotspot meiotic recombination.

Small ncRNAs (e.g., siRNA) base pair with their targets (RNA or DNA) and thereby deliver protein complexes to those targets [3], [4]. Similarly, ncRNA-DNA hybrids (R-loops) formed by long ncRNAs are implicated to direct the positioning of class switch recombination during B cell maturation [10], [49], [50]. We propose that an analogous mechanism operates to direct the machinery of meiotic recombination to ncRNA loci within chromosomes. This may involve a homology search of DNA by protein-RNA complexes (e.g., as is implicated for the siRNA-containing RITS complex). Alternatively, it may involve the recognition of R-loop structures by protein complexes (e.g., as is implicated for class switch recombination). In either case, the base pairing between ncRNA molecules and homologous chromosomal DNA would guide recombination to hotspots.

We emphasize that our proposals are not mutually exclusive with other hypothetical mechanisms proposed previously (reviewed by [12][14]) and the models even overlap to some extent. For example, R-loops would impart changes in local nucleosome organization and alter the sensitivity of DNA within chromatin to nucleases, which is a hallmark of most recombination hotspots and is invoked as a feature of most current models.

Implications and context

Our findings have two main implications. First, they reveal an unexpected, potentially unitary mechanism for what directs meiotic recombination to hotspots (expressed ncRNA loci). Second, they reveal a likely biological function for many of the enigmatic, polyadenylated ncRNA molecules that are so abundant in eukaryotes.

One of our two alternative hypotheses is that the polyadenylated ncRNA molecules help to position meiotic recombination. For context, this hypothesis is consistent with, and might explain mechanistically, some of the many connections between transcription and recombination documented previously (see Introduction). A few additional examples are provide here. Hotspot-activating RNA molecules would in principle confer differential DNA strand identity at hotspots, and hence could provide an underlying basis for the seemingly asymmetrical nature of all DSBs (directionality) [51] and for the preferential transfer of one DNA strand into heteroduplex DNA (strand identity) [52]. Similarly, our hypothesis may explain why the known RNA metabolism protein Ski8 (Rec14 of fission yeast) is an essential component of the Spo11 (Rec12) meiotic recombination protein complex in several organisms ([53][55] and our unpublished observations).

Materials and Methods

Relative proximity of DSB peaks to prl loci

Sixty-eight polyadenylated, ncRNA molecules of the prl class were identified among 987 random cDNA clones [5]. ORF [40], tos [38], and prl [5] locations were obtained from the fission yeast genome database at the Sanger institute [40]. Raw data on the genome-wide distribution of meiotic DSBs from dataset S1 [23] were analyzed with permission (Creative Commons Attribution License). Raw experimental data were averaged and divided by those of a negative, mitotic control to yield DSB intensity ratio, and peak curves for display were drawn using a sliding, 5-point average. Where available, we used the prl locations annotated in the Agilent microarray spreadsheet (Agilent Technologies). Seventeen prl loci were not annotated in that spreadsheet (prl7, prl9, prl11, prl15, prl17, prl18, prl22, prl25, prl27, prl29, prl30, prl49, prl56, prl61, prl63, prl64, plr67). These were added manually. One locus (prl52) was annotated twice (on chromosomes I and III). The position for chromosome I was used, as it is congruent with the location listed in the genome database. The distances between each prl locus and its two neighboring DSB peaks (left and right) were determined and tabulated in Excel 2005 (Microsoft Corporation, Redmond, WA). Ends of prl molecules (loci) that map within DSB peaks were assigned distance values of zero. Because 88% of the observed DSBs (from area integrals) were classified previously as falling within prominent DSB peaks [23], we analyzed the data both for prominent DSB peaks and for all DSB peaks (prominent plus weak) (Table 1).

Statistical measures

Each locus expressing an ncRNA molecule can map either within or outside of a DSB peak. Thus, under random genome-wide placement with constant per-locus “success” probability of mapping within a peak (the null hypothesis), the set of ncRNA loci should approximate the binomial distribution with respect to DSB peaks. We therefore modeled the data using the Binomial distribution. Binomial proportion parameters (success probabilities per locus) were estimated as equal to the fraction of the genome encompassed by “all” and “prominent” DSB peaks. Conditional binomial proportion parameters (success probabilities per locus, given that the locus is within an IGR) were similarly set equal to the fraction of IGRs encompassed by “all” and “prominent” DSB peaks. For every parameter estimate, the P value for the observed number of mappings to DSB peaks was calculated using the exact binomial test of a single proportion. Exact binomial one-sided upper confidence limits were also calculated for the expected number of successes under the null hypothesis. Calculations were performed using the “BINOMDIST” function in a Microsoft Excel spreadsheet (Microsoft Corporation, Redmond, WA). The binomial-proportion estimates, sample sizes, observed success rates, null-hypothesis confidence limits, and calculated P values for each test scenario are given in the main text.

Acknowledgments

We thank Giulia Baldini, Reine Protacio, and Alan Tackett for their input.

Author Contributions

Conceived and designed the experiments: WPW MKD. Performed the experiments: WPW MKD. Analyzed the data: WPW ERS MKD. Contributed reagents/materials/analysis tools: WPW ERS. Wrote the paper: WPW.

References

  1. 1. Nilsen TW (2007) RNA 1997–2007: a remarkable decade of discovery. Mol Cell 28: 715–720.TW Nilsen2007RNA 1997–2007: a remarkable decade of discovery.Mol Cell28715720
  2. 2. Mattick JS (2007) A new paradigm for developmental biology. J Exp Biol 210: 1526–1547.JS Mattick2007A new paradigm for developmental biology.J Exp Biol21015261547
  3. 3. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, et al. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305: 1437–1441.J. LiuMA CarmellFV RivasCG MarsdenJM Thomson2004Argonaute2 is the catalytic engine of mammalian RNAi.Science30514371441
  4. 4. Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, et al. (2004) RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303: 672–676.A. VerdelS. JiaS. GerberT. SugiyamaS. Gygi2004RNAi-mediated targeting of heterochromatin by the RITS complex.Science303672676
  5. 5. Watanabe T, Miyashita K, Saito TT, Nabeshima K, Nojima H (2002) Abundant poly(A)-bearing RNAs that lack open reading frames in Schizosaccharomyces pombe. DNA Res 9: 209–215.T. WatanabeK. MiyashitaTT SaitoK. NabeshimaH. Nojima2002Abundant poly(A)-bearing RNAs that lack open reading frames in Schizosaccharomyces pombe.DNA Res9209215
  6. 6. Griffiths-Jones S (2007) Annotating noncoding RNA genes. Annu Rev Genomics Hum Genet 8: 279–298.S. Griffiths-Jones2007Annotating noncoding RNA genes.Annu Rev Genomics Hum Genet8279298
  7. 7. Ng K, Pullirsch D, Leeb M, Wutz A (2007) Xist and the order of silencing. EMBO Rep 8: 34–39.K. NgD. PullirschM. LeebA. Wutz2007Xist and the order of silencing.EMBO Rep83439
  8. 8. Kasama T, Shigehisa A, Hirata A, Saito TT, Tougan T, et al. (2006) Spo5/Mug12, a putative meiosis-specific RNA-binding protein, is essential for meiotic progression and forms Mei2 dot-like nuclear foci. Eukaryot Cell 5: 1301–1313.T. KasamaA. ShigehisaA. HirataTT SaitoT. Tougan2006Spo5/Mug12, a putative meiosis-specific RNA-binding protein, is essential for meiotic progression and forms Mei2 dot-like nuclear foci.Eukaryot Cell513011313
  9. 9. Watanabe Y, Yamamoto M (1994) S. pombe mei2+ encodes an RNA-binding protein essential for premeiotic DNA synthesis and meiosis I, which cooperates with a novel RNA species meiRNA. Cell 78: 487–498.Y. WatanabeM. Yamamoto1994S. pombe mei2+ encodes an RNA-binding protein essential for premeiotic DNA synthesis and meiosis I, which cooperates with a novel RNA species meiRNA.Cell78487498
  10. 10. Yu K, Chedin F, Hsieh CL, Wilson TE, Lieber MR (2003) R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol 4: 442–451.K. YuF. ChedinCL HsiehTE WilsonMR Lieber2003R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells.Nat Immunol4442451
  11. 11. Page SL, Hawley RS (2003) Chromosome choreography: the meiotic ballet. Science 301: 785–789.SL PageRS Hawley2003Chromosome choreography: the meiotic ballet.Science301785789
  12. 12. Petes TD (2001) Meiotic recombination hot spots and cold spots. Nat Rev Genet 2: 360–369.TD Petes2001Meiotic recombination hot spots and cold spots.Nat Rev Genet2360369
  13. 13. Wahls WP (1998) Meiotic recombination hotspots: Shaping the genome and insights into hypervariable minisatellite DNA change. Curr Topics Dev Biol 37: 37–75.WP Wahls1998Meiotic recombination hotspots: Shaping the genome and insights into hypervariable minisatellite DNA change.Curr Topics Dev Biol373775
  14. 14. Nishant KT, Rao MR (2006) Molecular features of meiotic recombination hot spots. Bioessays 28: 45–56.KT NishantMR Rao2006Molecular features of meiotic recombination hot spots.Bioessays284556
  15. 15. Wahls WP, Smith GR (1994) A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity. Genes Dev 8: 1693–1702.WP WahlsGR Smith1994A heteromeric protein that binds to a meiotic homologous recombination hot spot: correlation of binding and hot spot activity.Genes Dev816931702
  16. 16. Kon N, Krawchuk MD, Warren BG, Smith GR, Wahls WP (1997) Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in S. pombe. Proc Natl Acad Sci USA 94: 13765–13770.N. KonMD KrawchukBG WarrenGR SmithWP Wahls1997Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in S. pombe.Proc Natl Acad Sci USA941376513770
  17. 17. Gao J, Davidson MK, Wahls WP (2008) Distinct regions of ATF/CREB proteins Atf1 and Pcr1 control recombination hotspot ade6-M26 and the osmotic stress response. Nucleic Acids Res 36: 2838–2851.J. GaoMK DavidsonWP Wahls2008Distinct regions of ATF/CREB proteins Atf1 and Pcr1 control recombination hotspot ade6-M26 and the osmotic stress response.Nucleic Acids Res3628382851
  18. 18. Schuchert P, Langsford M, Kaslin E, Kohli J (1991) A specific DNA sequence is required for high frequency of recombination in the ade6 gene of fission yeast. EMBO J 10: 2157–2163.P. SchuchertM. LangsfordE. KaslinJ. Kohli1991A specific DNA sequence is required for high frequency of recombination in the ade6 gene of fission yeast.EMBO J1021572163
  19. 19. Schultes NP, Szostak JW (1991) A poly(dA.dT) tract is a component of the recombination initiation site at the ARG4 locus in Saccharomyces cerevisiae. Mol Cell Biol 11: 322–328.NP SchultesJW Szostak1991A poly(dA.dT) tract is a component of the recombination initiation site at the ARG4 locus in Saccharomyces cerevisiae.Mol Cell Biol11322328
  20. 20. Steiner WW, Smith GR (2005) Natural meiotic recombination hot spots in the Schizosaccharomyces pombe genome successfully predicted from the simple sequence motif M26. Mol Cell Biol 25: 9054–9062.WW SteinerGR Smith2005Natural meiotic recombination hot spots in the Schizosaccharomyces pombe genome successfully predicted from the simple sequence motif M26.Mol Cell Biol2590549062
  21. 21. White MA, Wierdl M, Detloff P, Petes TD (1991) DNA-binding protein RAP1 stimulates meiotic recombination at the HIS4 locus in yeast. Proc Natl Acad Sci USA 88: 9755–9759.MA WhiteM. WierdlP. DetloffTD Petes1991DNA-binding protein RAP1 stimulates meiotic recombination at the HIS4 locus in yeast.Proc Natl Acad Sci USA8897559759
  22. 22. White MA, Dominska M, Petes TD (1993) Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 90: 6621–6625.MA WhiteM. DominskaTD Petes1993Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae.Proc Natl Acad Sci USA9066216625
  23. 23. Cromie GA, Hyppa RW, Cam HP, Farah JA, Grewal SI, et al. (2007) A discrete class of intergenic DNA dictates meiotic DNA break hotspots in fission yeast. PLoS Genet 3: e141.GA CromieRW HyppaHP CamJA FarahSI Grewal2007A discrete class of intergenic DNA dictates meiotic DNA break hotspots in fission yeast.PLoS Genet3e141
  24. 24. Gerton JL, DeRisi J, Shroff R, Lichten M, Brown PO, et al. (2000) Inaugural article: global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 97: 11383–11390.JL GertonJ. DeRisiR. ShroffM. LichtenPO Brown2000Inaugural article: global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae.Proc Natl Acad Sci USA971138311390
  25. 25. Yamada T, Mizuno KI, Hirota K, Kon N, Wahls WP, et al. (2004) Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot. EMBO J 23: 1792–1803.T. YamadaKI MizunoK. HirotaN. KonWP Wahls2004Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot.EMBO J2317921803
  26. 26. Shenkar R, Shen MH, Arnheim N (1991) DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot. Mol Cell Biol 11: 1813–1819.R. ShenkarMH ShenN. Arnheim1991DNase I-hypersensitive sites and transcription factor-binding motifs within the mouse E beta meiotic recombination hot spot.Mol Cell Biol1118131819
  27. 27. Ohta K, Shibata T, Nicolas A (1994) Changes in chromatin structure at recombination initiation sites during yeast meiosis. EMBO J 13: 5754–5763.K. OhtaT. ShibataA. Nicolas1994Changes in chromatin structure at recombination initiation sites during yeast meiosis.EMBO J1357545763
  28. 28. Wu TC, Lichten M (1994) Meiosis-induced double-strand break sites determined by yeast chromatin structure. Science 263: 515–518.TC WuM. Lichten1994Meiosis-induced double-strand break sites determined by yeast chromatin structure.Science263515518
  29. 29. Fan QQ, Petes TD (1996) Relationship between nuclease-hypersensitive sites and meiotic recombination hot spot activity at the HIS4 locus of Saccharomyces cerevisiae. Mol Cell Biol 16: 2037–2043.QQ FanTD Petes1996Relationship between nuclease-hypersensitive sites and meiotic recombination hot spot activity at the HIS4 locus of Saccharomyces cerevisiae.Mol Cell Biol1620372043
  30. 30. Myers S, Bottolo L, Freeman C, McVean G, Donnelly P (2005) A fine-scale map of recombination rates and hotspots across the human genome. Science 310: 321–324.S. MyersL. BottoloC. FreemanG. McVeanP. Donnelly2005A fine-scale map of recombination rates and hotspots across the human genome.Science310321324
  31. 31. Mizuno K, Koide T, Sagai T, Moriwaki K, Shiroishi T (1996) Molecular analysis of a recombinational hotspot adjacent to Lmp2 gene in the mouse MHC: fine location and chromatin structure. Mammalian Genome 7: 490–496.K. MizunoT. KoideT. SagaiK. MoriwakiT. Shiroishi1996Molecular analysis of a recombinational hotspot adjacent to Lmp2 gene in the mouse MHC: fine location and chromatin structure.Mammalian Genome7490496
  32. 32. Kon N, Schroeder SC, Krawchuk MD, Wahls WP (1998) Regulation of the Mts1-Mts2-dependent ade6-M26 meiotic recombination hotspot and developmental decisions by the Spc1 mitogen-activated protein kinase of fission yeast. Mol Cell Biol 18: 7575–7583.N. KonSC SchroederMD KrawchukWP Wahls1998Regulation of the Mts1-Mts2-dependent ade6-M26 meiotic recombination hotspot and developmental decisions by the Spc1 mitogen-activated protein kinase of fission yeast.Mol Cell Biol1875757583
  33. 33. Mieczkowski PA, Dominska M, Buck MJ, Lieb JD, Petes TD (2007) Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 104: 3955–3960.PA MieczkowskiM. DominskaMJ BuckJD LiebTD Petes2007Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in Saccharomyces cerevisiae.Proc Natl Acad Sci USA10439553960
  34. 34. Grimm C, Schaer P, Munz P, Kohli J (1991) The strong ADH1 promoter stimulates mitotic and meiotic recombination at the ADE6 gene of Schizosaccharomyces pombe. Mol Cell Biol 11: 289–298.C. GrimmP. SchaerP. MunzJ. Kohli1991The strong ADH1 promoter stimulates mitotic and meiotic recombination at the ADE6 gene of Schizosaccharomyces pombe.Mol Cell Biol11289298
  35. 35. Zahn-Zabal M, Lehmann E, Kohli J (1995) Hot spots of recombination in fission yeast: inactivation of the M26 hot spot by deletion of the ade6 promoter and the novel hotspot ura4-aim. Genetics 140: 469–478.M. Zahn-ZabalE. LehmannJ. Kohli1995Hot spots of recombination in fission yeast: inactivation of the M26 hot spot by deletion of the ade6 promoter and the novel hotspot ura4-aim.Genetics140469478
  36. 36. Keeney S, Giroux CN, Kleckner N (1997) Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88: 375–384.S. KeeneyCN GirouxN. Kleckner1997Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family.Cell88375384
  37. 37. Heckman DS, Geiser DM, Eidell BR, Stauffer RL, Kardos NL, et al. (2001) Molecular evidence for the early colonization of land by fungi and plants. Science 293: 1129–1133.DS HeckmanDM GeiserBR EidellRL StaufferNL Kardos2001Molecular evidence for the early colonization of land by fungi and plants.Science29311291133
  38. 38. Molnar M, Parisi S, Kakihara Y, Nojima H, Yamamoto A, et al. (2001) Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe. Genetics 157: 519–532.M. MolnarS. ParisiY. KakiharaH. NojimaA. Yamamoto2001Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe.Genetics157519532
  39. 39. Watanabe T, Miyashita K, Saito TT, Yoneki T, Kakihara Y, et al. (2001) Comprehensive isolation of meiosis-specific genes identifies novel proteins and unusual non-coding transcripts in Schizosaccharomyces pombe. Nucleic Acids Res 29: 2327–2337.T. WatanabeK. MiyashitaTT SaitoT. YonekiY. Kakihara2001Comprehensive isolation of meiosis-specific genes identifies novel proteins and unusual non-coding transcripts in Schizosaccharomyces pombe.Nucleic Acids Res2923272337
  40. 40. Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, et al. (2002) The genome sequence of Schizosaccharomyces pombe. Nature 415: 871–880.V. WoodR. GwilliamMA RajandreamM. LyneR. Lyne2002The genome sequence of Schizosaccharomyces pombe.Nature415871880
  41. 41. Nishant KT, Ravishankar H, Rao MR (2004) Characterization of a mouse recombination hot spot locus encoding a novel non-protein-coding RNA. Mol Cell Biol 24: 5620–5634.KT NishantH. RavishankarMR Rao2004Characterization of a mouse recombination hot spot locus encoding a novel non-protein-coding RNA.Mol Cell Biol2456205634
  42. 42. Cho YS, Iguchi N, Yang J, Handel MA, Hecht NB (2005) Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis. Biol Reprod 73: 840–847.YS ChoN. IguchiJ. YangMA HandelNB Hecht2005Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis.Biol Reprod73840847
  43. 43. Blitzblau HG, Bell GW, Rodriguez J, Bell SP, Hochwagen A (2007) Mapping of meiotic single-stranded DNA reveals double-stranded-break hotspots near centromeres and telomeres. Curr Biol 17: 2003–2012.HG BlitzblauGW BellJ. RodriguezSP BellA. Hochwagen2007Mapping of meiotic single-stranded DNA reveals double-stranded-break hotspots near centromeres and telomeres.Curr Biol1720032012
  44. 44. Buhler C, Borde V, Lichten M (2007) Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae. PLoS Biol 5: e324.C. BuhlerV. BordeM. Lichten2007Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae.PLoS Biol5e324
  45. 45. Hirota K, Steiner WW, Shibata T, Ohta K (2007) Multiple modes of chromatin configuration at natural meiotic recombination hot spots in fission yeast. Eukaryot Cell 6: 2072–2080.K. HirotaWW SteinerT. ShibataK. Ohta2007Multiple modes of chromatin configuration at natural meiotic recombination hot spots in fission yeast.Eukaryot Cell620722080
  46. 46. Hirota K, Hoffman CS, Shibata T, Ohta K (2003) Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot. Genetics 165: 505–515.K. HirotaCS HoffmanT. ShibataK. Ohta2003Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot.Genetics165505515
  47. 47. Maleki S, Keeney S (2004) Modifying histones and initiating meiotic recombination; new answers to an old question. Cell 118: 404–406.S. MalekiS. Keeney2004Modifying histones and initiating meiotic recombination; new answers to an old question.Cell118404406
  48. 48. Nicolas A (1998) Relationship between transcription and initiation of meiotic recombination: Toward chromatin accessibility. Proc Natl Acad Sci USA 95: 87–89.A. Nicolas1998Relationship between transcription and initiation of meiotic recombination: Toward chromatin accessibility.Proc Natl Acad Sci USA958789
  49. 49. Reaban ME, Griffin JA (1990) Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region. Nature 348: 342–344.ME ReabanJA Griffin1990Induction of RNA-stabilized DNA conformers by transcription of an immunoglobulin switch region.Nature348342344
  50. 50. Huang FT, Yu K, Balter BB, Selsing E, Oruc Z, et al. (2007) Sequence dependence of chromosomal R-loops at the immunoglobulin heavy-chain Smu class switch region. Mol Cell Biol 27: 5921–5932.FT HuangK. YuBB BalterE. SelsingZ. Oruc2007Sequence dependence of chromosomal R-loops at the immunoglobulin heavy-chain Smu class switch region.Mol Cell Biol2759215932
  51. 51. Neale MJ, Pan J, Keeney S (2005) Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436: 1053–1057.MJ NealeJ. PanS. Keeney2005Endonucleolytic processing of covalent protein-linked DNA double-strand breaks.Nature43610531057
  52. 52. Schar P, Kohli J (1994) Preferential strand transfer and hybrid DNA formation at the recombination hotspot ade6-M26 of Schizosaccharomyces pombe. EMBO J 13: 5212–5219.P. ScharJ. Kohli1994Preferential strand transfer and hybrid DNA formation at the recombination hotspot ade6-M26 of Schizosaccharomyces pombe.EMBO J1352125219
  53. 53. Arora C, Kee K, Maleki S, Keeney S (2004) Antiviral protein Ski8 is a direct partner of Spo11 in meiotic DNA break formation, independent of its cytoplasmic role in RNA metabolism. Mol Cell 13: 549–559.C. AroraK. KeeS. MalekiS. Keeney2004Antiviral protein Ski8 is a direct partner of Spo11 in meiotic DNA break formation, independent of its cytoplasmic role in RNA metabolism.Mol Cell13549559
  54. 54. Tesse S, Storlazzi A, Kleckner N, Gargano S, Zickler D (2003) Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition. Proc Natl Acad Sci USA 100: 12865–12870.S. TesseA. StorlazziN. KlecknerS. GarganoD. Zickler2003Localization and roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically distinct steps of meiotic homolog juxtaposition.Proc Natl Acad Sci USA1001286512870
  55. 55. Jolivet S, Vezon D, Froger N, Mercier R (2006) Non conservation of the meiotic function of the Ski8/Rec103 homolog in Arabidopsis. Genes Cells 11: 615–622.S. JolivetD. VezonN. FrogerR. Mercier2006Non conservation of the meiotic function of the Ski8/Rec103 homolog in Arabidopsis.Genes Cells11615622