RNA interference (RNAi) is an evolutionarily conserved defense mechanism to control expression of transposable elements and repetitive sequences in animals (FIRE et al. 1998; KETTING et al. 1999; TABARA et al. 1999). Double-stranded RNA (dsRNA) specifically degrades homologous mRNA in the cytoplasm (HAMMOND et al. 2001). The dsRNA involved in RNAi is produced in the nucleus or the cytoplasm by transcription through inverted DNA repeats, synthesis of sense and antisense RNAs, viral replication or viral RNA-dependent RNA polymerases (RdRP; MATZKE et al. 2001). Insight into the degradation process performed by dsRNAs comes from experiments in Drosophila. The dsRNA is first disrupted into sense and antisense strands with a length of 21- to 25 nucleotides. These small interference RNAs (siRNAs) are processed through an RNaseIII type protein, Dicer. Orthologs of Dicer have been identified in other organisms including mammals (TUSCHL et al. 1999;
ZAMORE et al. 2000). The antisense siRNAs generated by Dicer associate with a protein complex termed RISC (RNA-Induced Silencing Complex) and guides the complex to the complementary single stranded mRNA (HAMMOND et al. 2000). The mRNA-RISC complex is subsequently cut into two even pieces (fig. 5). Disrupted mRNA is further degraded (BERNSTEIN et al. 2001; ELBASHIR et al. 2001). It is possible that other pathways than that described here are also involved in the processing of siRNAs (MATZKE et al. 2001).
Genomes of higher eukaryotes contain a high number of parasitic sequences, transposable elements and endogenous viral sequences, which produce dsRNAs. It is thought that RNA interference has been evolved to counteract these dsRNAs (PLASTERK and KETTING 2000). The role of RNAi in development is not yet fully understood and only a few RNAi mutants showed developmental disorders such as failure in development of Arabidopsis or deregulation of developmental timing in Caenorhabditis elegans (VANCE and VAUCHERET 2001; PLASTERK and KETTING 2000). The contribution of RNA silencing by transcripts from transposable elements to the regulation of plant and animal development was also proposed and would be an explanation for the accumulation of these foreign sequences in the genomes of eukaryotes (MATZKE et al. 2000).
Figure 5: RNA interference (RNAi)
DICER possesses double stranded RNAs (dsRNAs) into small interference RNAs (siRNAs).
siRNAs associate with the protein RISK, which guides sense and antisense siRNAs to target single stranded mRNAs. Binding of siRNAs to their complementary mRNAs leads to denaturation of the mRNAs.
2.2 Genomic imprinting
Genomic imprinting is an epigenetic phenomenon and occurs in mammals and flowering plants. It is highly conserved among rodents, humans and ruminants (REIK and DEAN 2001; YOUNG et al. 2003). Around 0.1% of a mammalian genome is subject to imprinting. Expression of imprinted genes is restricted to one parental allele. DNA methylation at cytosine residues plays a predominant role in the mono-allelic gene expression of imprinted genes. Despite the functional non-equivalent role of the maternal and the paternal genomes due to different methylation patterns, both are required for normal mammalian development (BARTOLOMEI et al. 1991;
Double stranded RNA DICER
(RNaseIII type protein)
AAAAAAAAAA Discruption of complementary single stranded mRNA DICER cuts double stranded
RNA into small interference RNAs (siRNAs)
siRNAs associate with the protein complex RISC
RISC guides siRNAs to target mRNAs
AAAAAAAAAA Discruption of complementary single stranded mRNA DICER cuts double stranded
RNA into small interference RNAs (siRNAs)
siRNAs associate with the protein complex RISC
RISC guides siRNAs to target mRNAs
mammalian embryos, which possess a diploid maternal genome, are unable to develop to term. The same effect is observed when androgenetic embryos are transferred to recipient animals (McGRATH and SOLTER 1984b; SURANI et al.
1984; LOI et al. 1998; LAGUTINA et al. 2004). The first parthenogenetic mice were recently born and were generated from two haploid maternal genomes of non-growing and fully-grown oocytes that had been fused. The non-non-growing oocytes, which had been used as donor cells for nuclear transfer, contained a deletion of the imprinted H19 gene. This gene has been instrumental in unraveling the imprinting mechanism (KONO et al. 2004).
Less than 10% of all 5-methyl-cytosines (m5Cs) in the genome belong to imprinted genes and genes subject to X-inactivation in females (SMIT 1999). The majority of m5Cs are found in transposons (YODER et al. 1997). Methylation patterns of imprinted genes correlate with the parental allele from which they are inherited (SAPIENZA et al. 1987; REIK et al. 1987). This inherited methylation pattern is reversible in the next generation during germ cell development. A maternal allele could be a paternal allele in the next generation (REIK et al. 1987).
Mono-allelic gene expression can also be caused through polymorphic imprinting. This occurs when normally bi-allelically expressed genes are expressed from only one parental allele in a tissue specific manner due to allele specific base pair mutations. Polymorphic imprinting was reported for the WT1 gene (Wilms' tumor suppressor gene 1) and the PEG1/MEST genes (Paternally expressed gene 1/Mesodermal specific transcript) in humans (JINNO et al. 1994; PEDERSEN et al.
2002).
The list of imprinted genes is growing. To date, a variable number of imprinted genes has been identified among different species (Tab. 1; Imprinted gene catalogue: www.otago.ac.nz/IGC).
Mouse: 76 Human: 53 Sheep: 10 Pig: 2 Cattle: 7
Table 1: Imprinted genes identified in cattle (August 2004)
Imprinted loci
Chromosome Repressed
parental allele Name Reference
IGF2R/M6PR 9 Paternal
Insulin-like growth factor2
receptor
KILLIAN et al. 2001
Nnat 13 Paternal Neuronatin RUDDOCK et al.
20041
PEG3 18 Maternal
Paternally expressed
gene3
KIM et al. 2004
GTL2 21 Maternal Gene trap
locus2 DINDOT et al. 2004
IGF2 29 Maternal Insulin-like
growth factor2 DINDOT et al. 2004
Xist X Paternal X-inactive
specific transcript
DINDOT et al. 2004
H19 Paternal ZHANG et al. 2004
1 Imprinting of the Nnat gene was identified by qualitative analyses of mRNA abundance between in vitro fertilized and parthenogenetic bovine embryos. Determination of a SNP (single nucleotide