Download Detection of unpaired DNA at meiosis results in RNA‐mediated

What the papers say
Detection of unpaired DNA
at meiosis results in
RNA-mediated silencing
Michael J. Hynes* and Richard B. Todd
Summary
During meiosis, homologous chromosomes must pair in
order to permit recombination and correct chromosome
segregation to occur. Two recent papers(1,2) show that
meiotic pairing is also important for correct gene expression during meiosis. They describe data for the filamentous fungus Neurospora crassa that show that a lack of
pairing generated by ectopic integration of genes can
result in silencing of genes expressed during meiosis.
This can result in aberrant meioses whose defects are
specific to the function of the unpaired gene. Furthermore, mutations affecting the silencing mechanism have
been selected in a gene encoding a putative RNAdependent RNA polymerase. This finding indicates the
involvement of a meiotic specific post-transcriptional
gene silencing mechanism (PTGS) similar to that observed in vegetative cells in N. crassa and other organisms. Finally, this gene product is essential for normal
meiosis, suggesting that RNA-dependent processes are
fundamental to the sexual cycle. BioEssays 25:99–103,
2003. ß 2003 Wiley Periodicals, Inc.
Introduction
The involvement of small noncoding RNA molecules in a
variety of biological processes has recently excited considerable attention. Amongst these are microRNAs (miRNAs),
about 22–25 nucleotides long, generated from longer precursor molecules by the action of the Dicer class of
enzymes.(3–5) These are generally involved in regulating
mRNA stability and translation and include molecules (small
Department of Genetics, University of Melbourne, Australia.
Funding agency: Sequencing of A. fumigatus was funded by the
National Institute of Allergy and Infectious Disease U01 AI 48830 to D.
Denning and W. Nierman.
*Correspondence to: Prof. Michael J. Hynes, Department of Genetics,
University of Melbourne, Parkville 3010, Australia.
E-mail: [email protected]
DOI 10.1002/bies.10241
Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations: PTGS, post-transcriptional gene silencing; miRNAs,
microRNAs; stRNAs, small temporal RNAs; siRNAs, small intermediate RNAs; RdRP, RNA-dependent RNA polymerase; RIP, Repeat
Induced Point mutation; MSUD, Meiotic Silencing by Unpaired DNA.
BioEssays 25:99– 103, ß 2003 Wiley Periodicals, Inc.
temporal RNAs—stRNAs) that are involved in normal control
of development as exemplified by the lin4 and let7 genes of
Caenorhabditis elegans.(6–9) The other general class are the
small intermediate RNAs (siRNAs), derived from abnormal
RNA, that lead to targetted mRNA destruction (post-transcriptional gene silencing- PTGS) in response to sensing foreign
nucleic acids. A common theme of such silencing events
appears to be the synthesis of double stranded RNA by RNAdependent RNA polymerase (RdRP).(10,11) The fungus Neurospora crassa apparently has an intense dislike for aberrant
or duplicated nucleic acids and has two well-characterised
mechanisms to regulate expression of foreign or duplicated
sequences.(10,12) Studies of these mechanisms have made
significant contributions to the field of gene silencing. Two
recent papers describe a novel mechanism that senses
unpaired DNA at meiosis resulting in silencing of genes
expressed during meiosis.(1,2) If the gene is required for a
meiotic event then meiosis will halt at a characteristic stage.
Silencing in Neurospora crassa
N. crassa provides an excellent system for studying the
genetics, cytology and cell biology of meiosis.(13) Mating
occurs between strains carrying alternate mating type alleles
mat A and mat a, which are dissimilar in sequence
(idiomorphs),(14) and results in the formation of premeiotic
ascogenous hyphae in which nuclei fuse to form a diploid
nucleus (karyogamy). This is followed by immediate meiotic
divisions and one mitosis to yield eight nuclei, which are then
enclosed into ascospores linearily arranged in an ascus
(Fig. 1). The results of individual meioses can be observed
by ascospore pigmentation or morphology, and the fertility of
crosses assessed by ascospore yield and viability. In addition,
N. crassa is readily transformed by DNA carrying selectable
markers yielding transformants with DNA integrated either by
homology or ectopic integration at nonhomologous sites.
Two silencing mechanisms have previously been found in
this organism. One (Repeat-Induced Point mutation—RIP)
involves sensing duplicated sequences in the genome and
results in multiple C to T mutations in these sequences, in a
process specifically occurring in premeiotic cells.(12) Silencing,
therefore, is permanent and is observed only following
crossing. The second (quelling) detects multiple sequences
in the genome in vegetative hyphae (e.g. those generated by
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What the papers say
Figure 1. Silencing mechanisms occur in Neurospora crassa at
various stages of the life cycle. Neurospora crassa grows as
haploid multinucleate vegetative hyphae (A) Silencing of sequences present in multiple copies occurs in vegetative tissue by
Quelling. Mating occurs only between two individuals of opposing
mating type (mat A or mat a). Each individual can act as the female
and can be fertilized by male elements of the other strain. The
haploid nuclei derived from each strain proliferate in premeiotic
dikaryotic ascogenous hyphae (B) by conjugated nuclear divisions. It is within this dikaryotic tissue that duplicated sequences
are silenced by RIP (Repeat Induced Point-mutation). The hyphal
tip forms a crozier, the terminal pair of nuclei undergo one
synchronous mitotic division and the products are partitioned into
different cells such that the penultimate cell contains one nucleus
from each parent. These nuclei fuse (karyogamy) to form the
diploid zygote (C), which immediately undergoes meiosis. The
four meiotic progeny divide once mitotically and the resulting eight
nuclei are packaged into ascospores arranged linearly within an
ascus (D). During meiosis silencing of unpaired sequences occurs
by MSUD (meiotic silencing by unpaired DNA). MSUD is
observable in individual asci, with the specific phenotype
dependent on the meiotic or ascopore development function of
the silenced gene. Ascus development is accompanied by the
development of a fruiting body (perithecium) (E), which contains
all of the asci arising from a particular mating.
transforming DNA) and silences expression of these by
targetting the encoded mRNA for degradation.(10,15) The
isolation of quelling deficient mutants clearly indicates a
mechanism with similarities to PTGS mechanisms in other
systems involving 25 nucleotide long siRNA production from
aberrant transcripts and their amplification via RdRP.(16,17)
Meiotic silencing
The new mechanism was discovered via studies of a gene
(asm-1) required for mature melanized ascospore production.(18) Deletions of the gene are dominant resulting in all
ascospores in almost all asci being unmelanized and inviable.
Ectopic copies of a wild type gene cannot repair the defect
unless the cross is homozygous for the ectopic copy.(18) A
requirement for pairing suggested a mechanism formally
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related to the classical transvection phenomena of Drosophila.
However the finding that crosses with two paired and one
unpaired asm-1 copies result in virtually complete loss of
ascospore maturation indicates that it is the unpaired copy that
triggers silencing both of itself and the paired copies.(1)
Consequently this phenomenon of ascus dominance has
been termed Meiotic Silencing by Unpaired DNA (MSUD).
A number of other ascus-dominant mutations which affect
ascospore shape (Round spore), incorporation of nuclei into
ascospores (Banana) and linear ascospore arrangement
(Peak) also apparently result from MSUD.(1) In the latter case
most peak alleles are recessive but the one dominant allele
results from a translocation break point in the gene and hence
disrupts correct pairing. Furthermore studies with ectopic and
homologously integrated cloned genes such as the recA/
What the papers say
RAD51 orthologue and those encoding products predicted to
be required during meiotic divisions such as actin, b-tubulin
and histones clearly show MSUD resulting in characteristic
blocks in various stages of meiosis or spore maturation.(1) In
contrast, for metabolic genes essential during vegetative
growth but not during meiosis MSUD cannot be detected. It
can be noted that investigation of the specific MSUDgenerated block observed for meiotic genes of unknown
function provides a valuable tool for genetic dissection of
meiosis. The mat A and mat a alleles are not expected to pair
as they are dissimilar in sequence, and yet their expression is
required during meiosis. Therefore they are likely to be
immune to MSUD probably due to a special feature of their
flanking sequences. Consistent with this interpretation, matings between a mat a strain and one with mat A integrated
ectopically results in few ascospores.(1)
Mutations affecting silencing
Mutations affecting MSUD have been isolated(2) by selecting
for survivors of a cross between an Asm-1 deletion mutant and
a mutagenized strain with a wild type copy and an ectopic copy
of a frameshifted asm-1 mutant gene. These were individually
tested for suppression of MSUD by crossing to Round spore
and looking for normal ascospore shape in the progeny. An
initial mutant, Sad-1UV (suppressor of ascus dominance), was
found to result in about one third normal ascospores in a
heterozygous Round spore cross, with all eight spores in an
ascus being normal. Sad-1UV does not suppress the Round
spore phenotype itself because homozygous Round spore
crosses yield 100% abnormal spores. The general suppression of MSUD by Sad-1UV has been demonstrated by
suppression of the ascus dominance of Peak and Banana as
well as restoring fertility to crosses with unpaired ectopicallyintegrated actin, histone, b-tubulin, mating type and RecA/
RAD51 genes.(1) Strains with duplications of chromosomal
segments are barren, producing normal perithecia but no asci
(or few), in crosses to wild type strains. Heterozygous crosses
of Sad-1UV to such duplications showed increased fertility
were fertile(1) consistent with the hypothesis that MSUD resulting from unpaired meiotic genes in duplicated segments is at
least partly responsible for the dominant barren phenotype.
Furthermore, crosses between N. crassa and some closely
related species are infertile to varying degrees and Sad-1UV
was found to increase fertility in these interspecies crosses.(1)
This suggests that interspecies infertility at least in part results
from numerous chromosomal rearrangements leading to
lack of pairing of gene sequences that include genes essential
for meiosis. If MSUD is present in many organisms, then
this might constitute an important isolation mechanism for
speciation.
As selected, the Sad-1UV allele is partially dominant in
heterozygous crosses. The Sad-1UV allele is deleted for the 30
half of the gene and contains a few RIP mutations in the 50
half.(1) Homozygous Sad-1UV crosses are barren with all asci
arrested at meiotic prophase. This phenotype allowed mapping to linkage group I and cloning of sad-1þ by complementation.(2) It also has provided a more efficient method for the
isolation of further Sad-1 mutations. A complete sad-1 deletion
yielded almost 100% dominance while a series of sad-1 alleles
generated by the RIP procedure gave varying levels of
dominance correlating with both the number of induced
base-pair changes and amounts of DNA methylation throughout the sequence even though all were mutated in the start
codon.(1,2) DNA methylation is unlikely to be involved in
triggering MSUD since dim-2 or rid mutations, which result in a
lack of cytosine DNA-methyltransferases, do not suppress
MSUD.(2) The varying levels of dominance of sad-1 alleles in
heterozygous crosses can therefore be explained by autogenous MSUD leading to a low level of expression of sad-1þ
generated by lack of pairing at meiosis. This illustrates a use
for MSUD in analysing the stringency of requirements for
meiotic pairing by investigation of different alleles of genes
subject to MSUD at various locations in the genome.
The mechanism of meiotic
silencing—involvement of RNA
RT-PCR analysis of sad-1 transcripts revealed expression is
specific to sexual tissue and, furthermore, no induction by the
presence of unpaired DNA was observed in the crosses used
for RNA isolation.(2) sad-1 encodes a protein with significant
similarity to RNA-dependent RNA polymerases involved in
silencing in a variety of organisms, including QDE-1, a protein
essential for quelling in N. crassa.(2,19) Therefore, one may
conclude that synthesis of double stranded RNA and probably
RNA amplification is involved in MSUD, a step in common with
quelling and other PTGS mechanisms. It has become clear
that PTGS involves sensing aberrant RNA, formation of
dsRNA which is a substrate for the Dicer ribonuclease to
generate siRNAs which guide destruction of homologous
mRNA via a protein complex (RNA-induced silencing complex- RISC) containing another nuclease and are also used to
prime RdRP amplification to generate more siRNA via
DICER.(5,10,20) siRNA acts in trans to silence transcripts and
can be propagated between cells through vegetative tissue.(9,21–23) MSUD has been shown, using mosaic perithecia
arising from crosses involving a heterokaryotic female, to be
restricted to the ascus containing unpaired DNA.(2)
The story, to date, provokes several interesting questions.
For instance, how is the upstream signal for RNA-dependent
silencing detected? For quelling, N. crassa is able to detect
additional copies of normal genes. The signal for MSUD must
be unpaired meiotic DNA which could lead to the formation of
transcripts not normally synthesised because of pairing. Is any
unpaired region of a gene sufficient to trigger MSUD or is either
the 50 or 30 necessary? What other genes are involved in
MSUD? It seems likely that other components of the MSUD
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pathway will be conserved with the PTGS machinery.
Inspection of the N. crassa genome (Neurospora Sequencing
Project. Assembly version 3. Whitehead Institute/MIT Center
for Genome Research (http://www-genome.wi.mit.edu)) indicates the presence of dicer orthologs. Is one of these specific
to sexual development? Steps in the MSUD pathway must
play a normal essential role in meiosis since crosses homozygous for Sad-1 mutations are barren.(2) Therefore other
MSUD proteins may be involved in meiosis-perhaps a DICER
function generating one or more essential stRNAs. Genetic
dissection of MSUD via a comprehensive isolation of additional Sad genes will indicate whether MSUD and quelling use
common components and provide a unique opportunity for
analysing the requirements for pairing and its role in determining the progression of the meiotic cell cycle.
General implications
Silencing mechanisms identified in N. crassa are likely to play
a significant role in controlling invasion of the genome by
transposable elements and viruses.(24) RIP causes permanent gene inactivation and is probably of greatest importance
in resisting such invasions, as has been shown by studies of
the Tad-1 element in various N. crassa strains.(25) Quelling
will silence expression of transposon sequences present in
multiple copies restricting transposition in vegetative cells.
MSUD now adds a further mechanism for maintenance of
genome integrity as a cross between a strain containing one or
more transposons and one lacking such sequences will result
in unpaired meiotic DNA allowing MSUD to silence genes
required for transposition at perhaps a very vulnerable stage
for genome integration. The effects of Sad mutations on
transposition frequencies will test this proposal.
Just within the fungi interesting questions arise. Many fungi
have no known sexual cycle—the so-called Imperfect Fungi—
and it has been a long standing question whether this is because the correct strains or conditions for detection of mating
have not been discovered or whether, as indicated by population and evolutionary studies,(26–28) sexual reproduction has
been lost. The genome of Aspergillus fumigatus, a species
lacking a known sexual phase, contains clear orthologs of both
qde-1 and sad-1 (The Institute for Genomic Research, http://
www.tigr.org ). Since sad-1 expression is restricted to meiosis
this indicates that sex may yet be discovered in this species. A
less likely scenario is that in some species of fungi sad-1 may
play a role in vegetative cells. As more fungal genome sequences become available the extent of conservation of
silencing mechanisms will be of great interest.
The essential role of the MSUD pathway in meiosis raises
important questions regarding its generality. The special advantages of Neurospora for analysis of individual meioses permitted its discovery in the first place. Finding orthologs of Sad
genes in other species will allow their study in normal meiotic
processes as well as in the context of interspecies crosses.
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Acknowledgments
We thank David Perkins for useful comments on the manuscript. Neurospora crassa genome sequence data was
obtained from the Neurospora Sequencing Project, assembly
version 3, Whitehead Institute/MIT Center for Genome
Research (http://www-genome.wi.mit.edu). Preliminary Aspergillus fumigatus sequence data was obtained from
The Institute for Genomic Research website (http://www.
tigr.org).
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