An Argonaute-Like Protein Is Required for Meiotic Silencing

Corresponding author: Rodolfo Aramayo, College of Science, Texas A&M University, Rm. 415, Bldg. BSBW, College Station, TX 77843-3258., raramayo{at}mail.bio.tamu.edu (E-mail)

Communicating editor: M. E. ZOLAN

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

We demonstrate the involvement of suppressor of meiotic silencing-2 (sms-2⁺), a Neurospora gene coding for an Argonaute-like protein, in meiotic silencing and normal sexual development.

DURING meiosis, chromosomes "sense" each other through a process called meiotic transvection (ARAMAYO and METZENBERG 1996 ), which was discovered by studying the Ascospore maturation-1 (Asm-1) locus in Neurospora crassa (ARAMAYO and METZENBERG 1996 ; ARAMAYO et al. 1996 ). The presence of unpaired DNA was proposed to activate RNA silencing (SHIU et al. 2001 ) on the basis of the demonstration that mutations in an RNA-dependent RNA polymerase (RdRP) gene called Suppressor of ascus dominance-1 (Sad-1) eliminate the ascus dominance of unpaired DNA from Asm-1 and other genes (SHIU et al. 2001 ). Scanning of the Neurospora genome revealed the existence of a paralog for quelling deficient-2 (qde-2), which we call Suppressor of meiotic silencing-2 (Sms-2). The involvement of qde-2 and of the Sad-1 paralog [quelling deficient-1 (qde-1)] in the haploid vegetative RNA-silencing pathway called quelling has been firmly established (COGONI and MACINO 1997 ; CATALANOTTO et al. 2000 , CATALANOTTO et al. 2002 ). The identification of Sms-2 as a second gene related to qde-2 suggested its potential involvement in the meiotic silencing pathway. This is particularly attractive because SMS-2 belongs to the functionally novel, but highly conserved eukaryotic Argonaute protein family (Fig 1). We postulated that if Sms-2 is involved in meiotic silencing, Sms-2 loss-of-function mutants should suppress the ascus-dominant phenotype of unpaired Asm-1 DNA.

View larger version (50K): In this window In a new window Download PPT slide   Figure 1. Alignment of SMS-2-like proteins. (A) Histogram representation of the T-Coffee alignment (NOTREDAME et al. 2000 ) of SMS-2-related proteins. Histograms showing the consensus strength across all aligned proteins (All) and among only the six fungal proteins (Fungi) are shown (see Table S3, electronic supplementary material at http://www.genetics.org/supplemental/ for a complete description of the proteins, organisms, and identifiers). The strength of the alignment consensus as determined by MegAlign (DNASTAR, Madison, WI) using the PAM250 matrix correlates with the height of the bars. The tallest bars represent absolutely conserved positions in the alignment. The histograms were scaled to the alignment consensus sequence. Diagrammed below the histogram is the aligned SMS-2 protein with the PAZ and Piwi domains as determined by SMART (http://smart.embl-heidelberg.de/; SCHULTZ et al. 1998 ; LETUNIC et al. 2002 ; see full alignment in Figure S2, electronic supplementary material at http://www.genetics.org/supplemental/). (B) A Bayesian phylogenetic tree of SMS-2-related proteins created by MrBayes v2.01 (HUELSENBECK and RONQUIST 2001 ; HUELSENBECK et al. 2001 ) using the alignment represented in A. Branch lengths are scaled below the tree (see Figure S3, electronic supplementary material at http://www.genetics.org/supplemental/ for branch lengths and posterior probability values for the clades). Note that the fungal proteins cluster into two clearly distinguishable clades that we argue represent vegetative (quelling) and developmental (meiotic silencing) pathways in fungi (GALAGAN et al. 2003 ). Intriguingly, the animal proteins also cluster into two distinguishable clades, perhaps signifying two functional classes of Argonaute proteins in animals.

To test this hypothesis, we constructed two different null alleles of Sms-2 and tested their ability to suppress meiotic silencing. sms-2⁺ was mutagenized using repeat induced point mutation (RIP), generating two probable null alleles that we called Sms-2^RIP2 and Sms-2^RIP88. RIP occurs prior to meiosis when a sequence in Neurospora is present in more than one copy in the haploid genome (SELKER 1990 ). The Sms-2^RIP2 allele was generated by duplicating a 2374-bp DNA region corresponding only to the coding region of the gene, whereas the Sms-2^RIP88 was generated by duplicating the entire 5534-bp DNA region encompassing the upstream, coding, and downstream regions of Sms-2. In both cases the regions were duplicated by integration at the histidine-3 (his-3) locus in linkage group I (LG I). Sequencing of these alleles revealed, among many GC-to-AT transition mutations typical of RIP (see Figure S1, electronic supplementary material at http://www.genetics.org/supplemental), a CAG-to-TAG mutation changing a glutamine to a stop signal at position 118, thus potentially allowing the synthesis of a short truncated polypeptide from the Sms-2^RIP2 allele and an ATG-to-ATA mutation in the translation start codon of Sms-2^RIP88, thus blocking the synthesis of a complete polypeptide by this allele. As expected, these two alleles differ in the number of transition mutations present along their DNA regions. Most of the mutations present in Sms-2^RIP2 map only to the coding region of the gene. In contrast, the mutations present in Sms-2^RIP88 are distributed along the promoter, coding, and downstream regions of the allele.

The resulting Sms-2 mutants have no obvious defects during vegetative growth or asexual sporulation. In contrast, homozygous Sms-2^RIP2/Sms-2^RIP2 or Sms-2^RIP88/Sms-2^RIP88 crosses and heterozygous Sms-2^RIP2/Sms-2^RIP88 crosses are completely barren, as was demonstrated in homozygous crosses between Sad-1 loss-of-function alleles (SHIU et al. 2001 ). Similarly to Sad-1, heterozygous sms-2⁺/Sms-2^RIP2 and sms-2⁺/Sms-2^RIP88 crosses were fertile and produced normal ascospores (Table 1, crosses 1–5; Table 2; Table S2, electronic supplementary material at http://www.genetics.org/supplemental/).

The role of Argonautes in RNA silencing is not completely understood and may differ in the different systems studied (CARMELL et al. 2002 ). In Drosophila, Argonaute2 has been isolated as part of the RNA-inducing silencing complex (RISC; i.e., acting at the effector cycle, Fig 2; HAMMOND et al. 2001 ; HANNON 2002 ). In Caenorhabditis elegans, RDE-1 has been demonstrated to interact with RDE-4, a double-stranded RNA (dsRNA)-binding protein, and also with Dicer, an endonuclease (i.e., acting in the initiation step, Fig 2; HANNON 2002 ; TABARA et al. 2002 ). In Neurospora, testing the involvement of sms-2⁺ in meiotic silencing is not straightforward. The meiotic lethality observed in crosses involving homozygous loss-of-function Sms-2 alleles complicates the conceptually simple experiment of assaying meiotic silencing in the complete absence of sms-2⁺. The best-known experiments that can be done are to assay the degree of meiotic silencing in heterozygous sms-2⁺/Sms-2^RIP2 or sms-2⁺/Sms-2^RIP88 crosses (Fig 3). We predicted this to be possible because, according to the meiotic silencing model, in these crosses the inability of sms-2⁺ to pair with Sms-2^RIP2 or with Sms-2^RIP88 presumably induces partial silencing of the sms-2⁺ allele itself (Fig 3A). This cis-silencing is thus expected to significantly decrease the amounts of sms-2⁺ transcript and SMS-2 protein below that which is expected from a single fully functional gene. According to this logic, if there is enough active SMS-2 protein in heterozygous crosses (probably synthesized before the sms-2⁺ gene is silenced) to allow meiosis to proceed, but not enough to maintain a fully functional meiotic silencing machinery, we should be able to determine the involvement of sms-2⁺ in meiotic silencing.

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. Proposed meiotic silencing pathway of N. crassa: the proposed two steps and two cycles involved in initiating and maintaining meiotic silencing, respectively. Paired DNA does not induce the pathway. Unpaired DNA triggers the induction step, which involves the synthesis of aberrant RNA (aRNA) and its conversion to dsRNA by the SAD-1 RdRP. The presence of dsRNA triggers the initiation of the meiotic RNA-silencing process, which is composed of the following: the conversion of the dsRNA trigger into siRNAs (initiation step), predicted to occur via a Dicer-like endonuclease; the use of guide RNAs (gRNAs) as primers and single-stranded RNA (ssRNA) as template by SAD-1 RdRP to generate dsRNA (amplification cycle); and the incorporation of the gRNAs generated by both the initiation step and the amplification cycles into the RISC to direct the endonucleolytic cleavage of mRNA or ssRNA (effector cycle).

View larger version (30K): In this window In a new window Download PPT slide   Figure 3. Testing the role of sms-2+ in meiotic silencing in a diploid zygote cell. Boxed areas show the pairing of LG I, LG V, and LG VII chromosomes. Both LG V chromosomes carry wild-type alleles of Asm-1 (asm-1+). On LG I, one chromosome contains a DNA fragment corresponding to the Asm-1 region inserted at the his-3 locus, whereas the other chromosome carries no insert. On LG VII, one chromosome carries a wild-type allele of Sms-2 (sms-2+), whereas the other chromosome carries either a mutant allele of Sms-2 (Sms-2RIP; A and B) or a wild-type allele of Sms-2 (sms-2+; C). To the right is the schematic representation of a single ascus containing the predicted outcome from the different crosses. Inside these asci, solid ovals and open ovals represent viable and inviable ascospores, respectively.

To test this idea, we set up crosses heterozygous for Sms-2 (Fig 3A) and induced meiotic silencing by unpairing a copy of the reporter gene Asm-1. Silencing of Asm-1, whose gene product ASM-1 is required for ascospore maturation, results in white and inviable ascospores. We designed two strains; the first contained either Sms-2^RIP2 or Sms-2^RIP88 at the canonical locus in LG VII (Sms-2^RIP) in an otherwise wild-type background. The second strain contained Asm-1 DNA integrated at the his-3 locus in LG I (his-3⁺::Asm-1). Both strains contained wild-type asm-1⁺ alleles at their normal chromosomal positions in LG V (asm-1⁺). Because the unpaired Asm-1 region present in LG I (his-3⁺::Asm-1) has no pairing partner on the homologous chromosome, it is expected to trigger meiotic silencing, which, in turn, will silence all unpaired and paired copies of Asm-1 present in the genome. Under these conditions, if SMS-2 is part of the meiotic silencing machinery, a progeny of black and viable ascospores should be produced by these crosses despite the presence of an unpaired copy of Asm-1 (Fig 3A). In contrast, if SMS-2 is not part of the meiotic silencing machinery, the unpaired copy of Asm-1 (his-3⁺::Asm-1) will silence all Asm-1 copies present in the genome, resulting in a progeny of white and inviable ascospores (Fig 3B). In any case, control crosses homozygous for sms-2⁺ are expected to result in the production of a progeny of inviable ascospores (Fig 3C).

Progeny of viable ascospores were observed in crosses heterozygous for either Sms-2^RIP2 (crosses 6 and 7) or Sms-2^RIP88 (cross 8; Table 1). The percentage of mature ascospores observed in experimental crosses vs. the percentage observed in control crosses was 17.9% (cross 6) vs. 0.3% (cross 9), 30% (cross 7) vs. 0.3% (cross 10), and 23.2% (cross 8) vs. 0.3% (cross 10), respectively.

The persistence of a fraction of immature white ascospores, in our opinion, is at least partially attributable to RIP (SELKER 1990 ) and/or to an incomplete suppression (remember, these crosses are heterozygous for Sms-2). If RIP occurs in a gene that is essential for ascospore maturation, like Asm-1, spores carrying only the RIPed allele will not mature (ARAMAYO and METZENBERG 1996 ). In addition, if RIP occurs, the crippled silencing machinery present in the zygote of these heterozygous crosses is more capable of silencing the only remaining functional gene than of silencing the three functional genes that are present when RIP does not occur (D. W. LEE and R. ARAMAYO, unpublished results). Consistent with this interpretation, we observed a direct relationship between the insert size of the Asm-1 fragments used to induce silencing and the amount of black and viable ascospores produced (data not shown).

These results therefore establish the participation of Sms-2 in meiotic silencing and are consistent with qde-2 and Sms-2 functioning in two different RNA-silencing pathways (i.e., quelling and meiotic silencing, respectively). The biological roles of QDE-2 and SMS-2 proteins are neither redundant nor interchangeable on the basis of the fact that homozygous Sms-2^RIP2/Sms-2^RIP2 or Sms-2^RIP88/Sms-2^RIP88 crosses and heterozygous Sms-2^RIP2/Sms-2^RIP88 crosses, all homozygous for qde-2⁺, are completely barren and that expression of the qde-2⁺ coding region under the control of the Sms-2 promoter does not complement the meiotic barrenness of crosses homozygous for Sms-2 loss-of-function alleles (R. J. PRATT and R. ARAMAYO, unpublished results). In addition, homozygous qde-2 loss-of-function crosses undergo normal meiosis (R. J. PRATT and R. ARAMAYO, unpublished results). At the DNA level, Sms-2 is as related to an unrelated gene like Asm-1 as it is to qde-2 (data not shown), and at the protein level, SMS-2 is more identical to Argonaute-like proteins from Homo sapiens [e.g., 44.4% identity to brain-specific protein KIAA1567 (GenBank accession no. BAB13393.1)] than to QDE-2 (i.e., 37.7%; Fig 1B). On the basis of what we know, however, we still cannot exclude the possibility that genes like qde-2 play a minor role in meiotic silencing.

In contrast, demonstrating that Sad-1 and Sms-2 are both part of the same and only meiotic RNA-silencing pathway is not trivial, due to the nonlinear behavior of RNA-silencing pathways (HANNON 2002 ). On the basis of the hypothesis that proteins that function together in a pathway or structural complex are likely to evolve in a correlated fashion (MARCOTTE et al. 1999A , MARCOTTE et al. 1999B ; PELLEGRINI et al. 1999 ), we argue that Sad-1 and Sms-2 are both part of the same silencing pathway. This is because, in phylogenetic trees, SAD-1 and SMS-2 cluster consistently with their corresponding homologs of the single functional pathway observed in Schizosaccharomyces pombe (HALL et al. 2002 ; VOLPE et al. 2002 ) and GALAGAN et al. 2003 .

To study the genetic relationship between Sad-1 and Sms-2, we tested the meiotic behavior of single and double mutants. We generated a null allele of Sad-1 (Sad-1^RIP64) using RIP by duplicating a 2711-bp DNA region corresponding to the promoter and coding region of the gene at the his-3 locus in LG I (for details see Methods, electronic supplementary material at http://www.gentics.org/supplemental/). As predicted for a loss-of-function allele, homozygous Sad-1^RIP64/Sad-1^RIP64 crosses were completely barren, as was found in homozygous crosses between Sad-1 deletion alleles (SHIU et al. 2001 ; SHIU and METZENBERG 2002 ). Heterozygous sad-1⁺/Sad-1^RIP64 crosses produced ascospores, but their number was greatly reduced compared to the number generated in crosses between sad-1⁺ strains (K. BAKER and R. ARAMAYO, unpublished data). Therefore, as in the case of Sms-2, testing the involvement of sad-1⁺ in meiotic silencing in the complete absence of sad-1⁺ is not possible, and here again, performing heterozygous crosses is the best known way to go.

We first tested the ability of Sad-1^RIP64 to suppress meiotic silencing. For this we constructed strains containing Sad-1^RIP64 at the canonical locus in LG I in either an asm-1⁺ or an Asm-1 background [Asm-1^(3430-9336); Table 1]. In both cases, strains contained wild-type asm-1⁺ alleles at the ectopic his-3 chromosomal position in LG I. During meiosis, asm-1⁺ at its canonical position has no pairing partner due to the presence of the Asm-1^(3430-9336) deletion allele in the homologous chromosome. This is expected to trigger silencing, which, in turn, will silence all unpaired and paired copies of Asm-1 present in the genome. As expected, viable ascospore progeny were observed in crosses heterozygous for Sad-1^RIP64 (crosses 11–14, Table 1). The percentage of mature ascospores observed in reciprocal experimental crosses vs. the percentage observed in control crosses was 51.3% (cross 11) and 48.9% (cross 12) vs. 0% (cross 13) and 53.5% (cross 14) and 52% (cross 15) vs. 0.1% (cross 16), respectively. We then determined the fraction of mature ascospores produced by crosses heterozygous for Sms-2 (sms-2⁺/Sms-2^RIP88) and Sad-1, Sms-2 (sad-1⁺/Sad-1^RIP64, sms-2⁺/Sms-2^RIP88), to be 16.7% (cross 17) and 48.7% (cross 18; Table 1), respectively. The reduced percentage of mature ascospores observed in crosses between strains, each of them containing duplicated DNA (36.8%, cross 19, Table 1), in our opinion is attributable to RIP and to the consequent induction of meiotic silencing that results from the inability of the asm-1⁺ allele(s) to pair with their RIPed partners in their homologous chromosomes.

These results are consistent with the idea that Sad-1 and Sms-2 are both necessary but not sufficient for meiotic silencing. They also demonstrate that Sad-1 is genetically epistatic to Sms-2. In addition, under our working meiotic silencing model (Fig 2), it is expected that mutations in Sad-1 would have a more profound effect than mutations in Sms-2 in the functioning of the pathway. This is because mutations affecting Sad-1, whose gene product is predicted to act at two different stages within the pathway (Fig 2), are expected to stop the silencing reaction at an early stage. In contrast, mutations affecting Sms-2, whose gene product is predicted to act in the effector cycle (Fig 2), are not expected to affect the silencing mediated by the SAD-1-RdRP and Dicer in the initiation step (Fig 2). Two Dicer-like genes in the Neurospora genome could serve this function (GALAGAN et al. 2003 ). This interpretation is consistent with the accumulation of siRNA detected during quelling in qde-2 loss-of-function mutants (CATALANOTTO et al. 2002 ).

The connection between the presence of unpaired DNA and RNA silencing during meiosis is tantalizing. Every component of the meiotic silencing pathway identified to date is required for the completion of meiotic prophase (SHIU et al. 2001 ). Current dogma dictates that all RNA-based gene-silencing mechanisms share the objective of protecting the genome from invading transposable elements, but why would a meiotic RNA-silencing pathway be conserved in a genome that has evolved very efficient mechanisms for detecting repeated elements (e.g., RIP)? In our view, the products of the meiotic RNA-silencing machinery either play a previously unrecognized gene regulatory role during meiosis (e.g., via production of micro-RNAs; AMBROS 2001 ; LAGOS-QUINTANA et al. 2001 ; LAU et al. 2001 ; LEE and AMBROS 2001 ) or have evolved as key components of the meiotic chromosome biology (e.g., via formation of heterochromatin; BERNARD et al. 2001 ; ALLSHIRE 2002 ; DERNBURG and KARPEN 2002 ; JENUWEIN 2002 ; REINHART and BARTEL 2002 ; VOLPE et al. 2002 ).

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AF500110, AF508210, AF508211, and AF508212.

	ACKNOWLEDGMENTS

We thank Michael D. Manson, Debby Siegele, and Jim Hu for constant encouragement. R.J.P. was partially supported by the Program in Microbial Genetics and Genomics. This work was supported by U.S. Public Health Service grant GM58770 to R.A.

Manuscript received August 29, 2002; Accepted for publication February 5, 2003.

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				Q. Wang and G. G. Carmichael Effects of Length and Location on the Cellular Response to Double-Stranded RNA Microbiol. Mol. Biol. Rev., September 1, 2004; 68(3): 432 - 452. [Abstract] [Full Text] [PDF]

				D. W. Lee, K.-Y. Seong, R. J. Pratt, K. Baker, and R. Aramayo Properties of Unpaired DNA Required For Efficient Silencing in Neurospora crassa Genetics, May 1, 2004; 167(1): 131 - 150. [Abstract] [Full Text] [PDF]

				K. A. Borkovich, L. A. Alex, O. Yarden, M. Freitag, G. E. Turner, N. D. Read, S. Seiler, D. Bell-Pedersen, J. Paietta, N. Plesofsky, et al. Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism Microbiol. Mol. Biol. Rev., March 1, 2004; 68(1): 1 - 108. [Abstract] [Full Text] [PDF]

				M. PAL-BHADRA, U. BHADRA, and J.A. BIRCHLER Interrelationship of RNA Interference and Transcriptional Gene Silencing in Drosophila Cold Spring Harb Symp Quant Biol, January 1, 2004; 69(0): 433 - 438. [Abstract] [PDF]