Whole genome microarray analysis of C. elegans rrf-3 and eri-1 mutants

Department of Neurobiology, A.I. Virtanen Institute, Kuopio University, Kuopio 70211, Finland.
FEBS Letters (Impact Factor: 3.17). 10/2007; 581(26):5050-4. DOI: 10.1016/j.febslet.2007.09.043
Source: PubMed


We performed genome wide gene expression analysis on L4 stage Caenorhabditis elegans rrf-3(pk1426) and eri-1(mg366) mutant strains to study the effects caused by loss of their encoded proteins, which are required for the accumulation of endogenous secondary siRNAs. Mutant rrf-3 and eri-1 strains exhibited 72 transcripts that were co-over-expressed and 4 transcripts co-under-expressed (>2-fold, P<0.05) compared to N2 wild type strain. Ontology analysis indicated these transcripts were over represented for protein phosphorylation and sperm function genes. These results provide additional support for the hypothesis that RRF-3 and ERI-1 act together in the endo-siRNA pathway, and furthermore suggests their involvement in additional biological processes.

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    • "These roles for RRF-3 in endogenous gene regulation and in exo RNAi efficacy are shared with a group of factors in the ERI pathway (so named because of Enhanced RNAi phenotypes). These include the widely conserved exoribonuclease and DCR-1 complex member ERI-1, the uncharacterized Dicer complex member ERI-9, DCR-1 itself, and the Argonaute ERGO-1 (Asikainen et al., 2007; Duchaine et al., 2006; Gabel and Ruvkun, 2008; Gent et al., 2009; Kennedy et al., 2004; Pavelec et al., 2009). All of these factors appear to have somatic roles, as their activity in siRNA production modulates the subcellular localization of a somatically expressed Argonaute NRDE-3, and their mutations increase efficacy of exo RNAi against somatic genes. "
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    ABSTRACT: Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26 nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the Argonaute ERGO-1, DICER, and a series of associated ("ERI") factors. This primary process leads to production of a much more abundant class of 22 nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA.
    Molecular cell 03/2010; 37(5):679-89. DOI:10.1016/j.molcel.2010.01.012 · 14.02 Impact Factor
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    • "In such a case, either the target mRNA may be grossly overexpressed or the temporal expression may be precocious and/or persistent. In support of model 1, microarray analysis comparing wild-type animals to eri-1(À) or rrf-3(À) animals indicates that sperm RNAs are negatively regulated by ERI-1 and RRF-3 (Asikainen et al. 2007). We revisited the eri-1/rrf-3 microarray data sets and compared these data to existing microarray data sets, which identified genes preferentially expressed in the sperm (Reinke et al. 2000, 2004). "
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    ABSTRACT: Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(-) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.
    Genetics 10/2009; 183(4):1283-95. DOI:10.1534/genetics.109.108134 · 5.96 Impact Factor
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    • "By comparing whole males either mutant or wild type for rrf-3, we have identified exemplary cellular mRNAs for which siRNA production during spermatogenesis depends on RRF-3. These cellular mRNAs show strong enrichment for previously identified genes whose mRNA levels are elevated in populations of whole animals in both rrf-3 and eri-1 mutants (Asikainen et al. 2007). RRF-3 and paternal contributions to embryogenesis: "
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    ABSTRACT: Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing through formation of RNA duplexes. Although progress has been made in identifying the capabilities of siRNAs in silencing foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a Caenorhabditis elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. In a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role of the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis.
    Genetics 10/2009; 183(4):1297-314. DOI:10.1534/genetics.109.109686 · 5.96 Impact Factor
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