Trends in Genetics
Volume 20, Issue 9, September 2004, Pages 417-423
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RIP: the evolutionary cost of genome defense

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Repeat-induced point mutation (RIP) is a homology-based process that mutates repetitive DNA and frequently leads to epigenetic silencing of the mutated sequences through DNA methylation. Consistent with the hypothesis that RIP serves to control selfish DNA, an analysis of the Neurospora crassa genome sequence reveals a complete absence of intact mobile elements. As in most eukaryotes, the centromeric regions of N. crassa are rich in sequences that are related to transposable elements; however, in N crassa these sequences have been heavily mutated. The analysis of the N. crassa genome sequence also reveals that RIP has impacted genome evolution significantly through gene duplication, which is considered to be crucial for the evolution of new functions. Most if not all paralogs in N. crassa duplicated and diverged before the emergence of RIP. Thus, RIP illustrates the extraordinary extent to which genomes will go to defend themselves against mobile genetic elements.

Section snippets

What is RIP?

RIP is a process that efficiently detects and mutates duplicated sequences 2, 5 (Figure 1). Acting only during the sexual cycle, RIP identifies duplications that are greater than ∼400bp (or ∼1kb in the case of unlinked duplications) [6] and introduces C:G to T:A mutations into both copies of the duplicated sequences. All duplications that share greater than ∼80% nucleotide identity [7] are detected efficiently and in a single passage through the sexual cycle, up to ∼30% of the C:G pairs in

Evolution by gene duplication

One of the most striking characteristics of the N. crassa genome sequence is the almost complete absence of highly similar gene pairs (Figure 2). Of the predicted 10 082 N. crassa protein-coding genes, only six pairs (12 genes) share >80% nucleotide or amino-acid identities in their coding sequences (Figure 2). This value is significant because, as described previously, RIP mutates duplicated sequences that share greater than ∼80% nucleotide similarity. Five of the six pairs of highly similar

RIP and mobile elements

The deactivation of repeated sequences by RIP serves as an effective defense against mobile elements. Early studies revealed clear evidence of inactivation by RIP of both the retrotransposon Tad (transposon from Adiopodoumé) [17] and the DNA-type transposon Punt (putative transposon) [15], consistent with failures to detect transpositions in numerous wild Neurospora strains. An analysis of sequences from the centromeric region of linkage group VII revealed the relics of several additional types

Repeated genes persisting in the face of RIP

Only a few repeated genes are known to survive RIP. The dispersed 5S rRNA and tRNA genes apparently evade RIP because their length is below the threshold for RIP; indeed, the first known natural relic of RIP consists of a tandem duplication of an 800 bp segment that includes a 5S rRNA gene [9]. The only sizeable repetitive sequences that are known to persist in spite of RIP in N. crassa are ∼175 copies [21] of the tandemly arranged, 9 kb rDNA repeats that give rise to the 17S, 5.8S and 25S rRNAs.

Mechanism of RIP

The study of the mechanism of RIP by biochemical approaches is difficult owing to the microscopic ascogenous tissue in which the process takes place. In addition, classical genetic studies of RIP are hampered by the fact that the cells in this tissue contain nuclei from each parent, preventing recognition of recessive defects. As a result, only one component of the molecular machinery for RIP has been reported to date. A candidate-gene approach was used to identify a gene required for RIP

Relationship between RIP and methylation

Methylation in N. crassa appears to be closely related to RIP. Approximately 2% of cytosines in the N. crassa genome are methylated [36] and, as with animals and plants, methylation has been shown to cause gene silencing in this fungus [11]. In contrast to mammals and plants, methylation in N. crassa is not biased towards symmetric (e.g. CpG) sites [34]. DNA methylation is typically heavy but heterogeneous, with every cytosine in the affected region having a >80% chance of being methylated [35]

Phylogenetic distribution

Since its discovery in N. crassa, evidence has accumulated that RIP or similar processes occur in other fungi. Experimental evidence of repeat-induced mutation has been reported in Magnaporthe grisea 43, 44, Podospora anserina [45] and Leptosphaeria maculans [46] (Table 1). In addition, transposons displaying mutations that are consistent with RIP have been identified in various other fungi including several Neurospora species [17], in addition to Fusarium oxysporum 47, 48, 49, Aspergillus

Perspectives and conclusions

The nature of RIP and its impact on the N. crassa genome raise several interesting considerations. Gene duplication has been considered the primary means by which organisms evolve new genes [14]. Although there has been considerable debate over the degree of selection acting on duplicated genes, the frequency with which beneficial mutations are generated, the rate at which new functions evolve and the rate at which duplicated genes are lost 57, 58, gene duplication has nonetheless remained

Acknowledgements

We thank Michael Freitag, Kristina Smith, Sarah Calvo and Bruce Birren for their feedback and comments. This work was supported by NIH grants GM35690 (E.U.S.), HG02045–05 (J.E.G.) and HG02152–03 (J.E.G), and by NSF grants MCB-0131383 (E.U.S.) and 0078148 (J.E.G.).

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